Methods and compositions for degrading pectin

ABSTRACT

The present invention provides enriched polynucleotides, and enriched polypeptides having pectinase activity. The present invention also includes methods of using the polynucleotides and polypeptides described herein. For instance, the methods include producing a metabolic product, such as ethanol.

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Ser.No. 61/097,975, filed Sep. 18, 2008, and 61/179,570, filed May 19, 2009,each of which is incorporated by reference herein.

BACKGROUND

The US Energy Independence and Security Act (EISA) of 2007 states thattransportation fuel introduced into commerce in the US (annual average)contain at least 12.95 billion gallons of renewable fuels by the year2010. Ethanol is the most prevalent renewable fuel, with the USproducing over 6 billion gallons in 2007 (Peterson and Ingram, 2008,Ann. N.Y. Acad. Sci., 1125:363-372) and 9 billion gallons in 2009.Currently, the majority of ethanol is produced from corn; however,limited supply will force ethanol production from other sources ofbiomass, of which the US produces over a billion tons annually—enough toproduce 80 billion gallons of renewable fuel (Gray et al., 2006,Bioethanol. Curr. Opin. Chem. Biol., 10:141). Moreover, use of wastebiomass for fuel production positively affects greenhouse gases andcarbon debt without causing land-use change (Fargione et al., 2008,Science, 319:1235-1237, Searchinger et al., 2008, Science,319:1238-1240). The EISA of 2007 also requires that ethanol, and otherliquid transportation fuels such as butanol and biomass-based diesel,derived from any of the following: cellulose, hemicellulose, lignin,sugar, starch (other than corn starch), waste material and residues, beincorporated into our 2010 fuel supply at 0.95 billion gallons. By theyear 2015 over 5 billion gallons of advanced renewable fuel from biomassother than corn starch are required to be available for use in ourtransportation sector.

Unlike corn grain, where the major component is starch, other sources ofbiomass are composed of 40-50% cellulose, 25-35% hemicellulose, and15-20% lignin (Gray et al., 2006, Bioethanol. Curr. Opin. Chem. Biol.,10:141, Doran-Peterson et al., 2008, The Plant J., 54:582-592). Thehighly complex biomass structure has necessitated development of manyprocesses for fuel ethanol conversions from substrates containinglignocellulose, which can include thermochemical and/or mechanicalpretreatment to allow enzymatic access, enzymatic degradation to reducesubstrates to fermentable sugars, and finally fermentation of thosesugars by microorganisms. Commercially available enzyme mixtures areusually culture supernatants from fungi, and sometimes bacteria,containing a complex of enzyme activities. In order to efficientlydegrade cellulose several major classes of enzymes are required, such asendo-β-1,4-glucanases (endocellulase, Cx—cellulase; EC 3.2.1.4) whichcleave internal β-1,4-glycosidic bonds generating oligosaccharides;exo-β-1,4-glucanases (exocellulase, cellobiohydrolaseC1-cellulase; EC3.2.1.91) which cleave the non-reducing end to release a dimer ofglucose called cellobiose; and β-glucosidase (cellobiase, EC 3.2.1.21)which cleaves cellobiose into monomeric glucose molecules (Whitaker,1994, Principles of Enzymology for the Food Sciences, 2nd Ed. MarcelDekker, New York, Gilkes et al., 1991, Microbiol Rev., 55:303-315,Henrissat et al., 1989, Gene, 81(1):83-95, Beguin et al., 1994, FEMSMicrobiol Rev., 1994 13(1):25-58). Many commercial preparations aredeficient in cellobiase, and when this dissacharide accumulates it caninhibit further enzyme deconstruction of the cellulose microfibrils.

In some biomass types, such as sugar beet pulp and citrus peel, pectincan also compose a significant portion of the lignocellulose structureand functions as a matrix to hold cellulose and hemicellulose fibers.The pectin backbone can consist of a homopolymer of α-1,4-D-galacturonicacid (homogalacturonan) or repeats of the disaccharideα-1,2-L-rhamnose-α-1,4-D-galacturonic acid (rhamnogalacturonan-I), and,typically, 70% to 80% of galacturonic acid residues are methylated.Homogalacturonan can be substituted with xylose or apiose, whilerhamnogalacturonan-I is often substituted with galactose, arabinose, orgalactan (Willats et al., 2001, Plant Mol. Biol., 47:9-27, Ridley etal., 2001, Phytochemistry, 57:929-967).

The degradation of pectin requires both methylesterases anddepolymerases. Pectin methylesterases are responsible for the hydrolysisof methylester linkages from the polygalacturonic acid backbone(Whitaker, 1984, Enzyme Microbial Technol., 6:341-347). Pectindepolymerases act upon the polygalacturonate backbone and belong to oneof two families: polygalacturonases or lyases. Polygalacturonases areresponsible for the hydrolytic cleavage of the polygalacturonate chain,while lyases cleave by β-elimination giving a Δ4,5-unsaturated product(Jayani et al., 2005, Process Biochem., 40:2931-2944, Sakai et al.,1993, Adv. Appl. Microbiol., 39:231-294). There are two types of lyases:pectate lyases, which cleave unesterified polygalacturonate, or pectate;and pectin lyases, which cleave methyl esterified pectin. Pectate lyaseshave been classified into families based on amino acid similarity, whichin turn suggests structural features (Coutinho and Henrissat, 1999, In:Gilbert et al. (Eds.) Recent Advances in Carbohydrate Bioengineering.Cambridge, The Royal Society of Chemistry).

Once the lignocellulosic biomass is degraded into fermentable sugars,many different types of sugars, including pentose and acidic sugars areliberated for metabolism to a product(s) (Doran-Peterson et al., 2008,The Plant J., 54:582-592). Most ethanol fermentations in the U.S. todayuse the yeast Saccharomyces cerevisiae to convert starch glucose intoethanol and CO₂; however, lignocellulosic biomass contains many sugarsthat S. cerevisiae is unable to ferment (Peterson and Ingram, 2008, Ann.N.Y. Acad. Sci., 1125:363-372). Thus, Escherichia coli, which is capableof using these hexoses and pentoses, was engineered as a biocatalyst forethanol production by integration of the pyruvate decarboxylase (pdc)and alcohol dehydrogenase II (adhB) genes from Zymomonas mobilis intothe chromosome of E. coli to generate strain K011 (Ohta et al., 1991,Appl. Environ. Microbiol., 57:893-900).

SUMMARY OF THE INVENTION

Provided herein are polynucleotides that may be enriched, isolated, orpurified. The polynucleotides include (a) a nucleotide sequence encodinga polypeptide having pectinase activity, wherein the amino acid sequenceof the polypeptide and the amino acid sequence of SEQ ID NO:4 have atleast 80% identity, (b) a nucleotide sequence encoding a polypeptidehaving pectinase activity, wherein the nucleotide sequence of theisolated polynucleotide and the nucleotide sequence of SEQ ID NO:3 haveat least 80% identity, (c) a nucleotide sequence encoding a polypeptidehaving pectinase activity, wherein the amino acid sequence of thepolypeptide and the amino acid sequence of SEQ ID NO:2 have at least 80%identity, and (d) a nucleotide sequence encoding a polypeptide havingpectinase activity, wherein the nucleotide sequence of the isolatedpolynucleotide and the nucleotide sequence of SEQ ID NO:1 have at least80% identity. Also disclosed are the full complements of the nucleotidesequences. The polynucleotide may be operably linked to at least oneregulatory sequence, and may further include heterologous nucleotides.The polynucleotide may be part of a vector. Also disclosed aregenetically modified microbes that include an exogenous polynucleotidedescribed herein.

Also provided are polypeptides that may be enriched, isolated, orpurified. The polypeptides have pectinase activity. The polypeptidesinclude an amino acid sequence, wherein the amino acid sequence and theamino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 80%identity. The polypeptide may be an exogenous polypeptide present in agenetically modified microbe, such as a gram-negative microbe (e.g., E.coli) or a fungus (e.g., S. cerevisiae). The genetically modifiedmicrobe may also include a polynucleotide encoding a polypeptide havingoligogalacturonate activity. Further provided are compositions thatinclude the polynucleotides and/or the polypeptides described herein, aswell as compositions that include the genetically modified microbesdescribed herein.

Yet further provided are methods for using the polynucleotides and/orpolypeptides described herein. Methods for degrading pectin may includecontacting a composition that contains pectin with a polypeptide havingpectinase activity and disclosed herein under conditions suitable forthe degradation of the pectin. The polypeptide used in the method may beenriched, isolated, or purified. The polypeptide may be expressed by agenetically modified microbe, and the contacting may include contactingthe pectin with the genetically modified microbe. The geneticallymodified microbe may produce a metabolic product, such as ethanol, andthe method may further include recovering the metabolic product. Thecomposition may include a lignocellulosic material that is obtainedfrom, for instance, a fruit or a vegetable. The pectin may be esterifiedor unesterified. If esterified, the level of esterification may be atleast 8.5%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, or at least 80%. The method may further includecontacting the degraded pectin with a polypeptide havingoligogalacturonate activity.

Also provided are methods for producing a metabolic product. The methodsmay include contacting a composition containing pectin with agenetically modified microbe under conditions suitable for thedegradation of the pectin, wherein the genetically modified microbeincludes a polypeptide having pectinase activity and disclosed herein.The method may further include contacting the degraded pectin with apolypeptide having oligogalacturonate activity. In another embodiment,the methods may include contacting a composition that contains pectinwith a genetically modified microbe under conditions suitable for thedegradation of the pectin, wherein the genetically engineered microbecomprises an exogenous polypeptide having pectinase activity and anexogenous polypeptide having oligogalacturonate activity. The metabolicproduct may be ethanol, and the method may further include recoveringthe metabolic product. The composition may include a lignocellulosicmaterial that is obtained from, for instance, a fruit or a vegetable.The pectin may be esterified or unesterified. If esterified, the levelof esterification may be at least 8.5%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, or at least 80%.

As used herein, the term “polynucleotide” refers to a polymeric form ofnucleotides of any length, either ribonucleotides or deoxynucleotides,and includes both double- and single-stranded RNA and DNA. Apolynucleotide can be obtained directly from a natural source, or can beprepared with the aid of recombinant, enzymatic, or chemical techniques.A polynucleotide can be linear or circular in topology. A polynucleotidemay be, for example, a portion of a vector, such as an expression orcloning vector, or a fragment. A polynucleotide may include nucleotidesequences having different functions, including, for instance, codingregions, and non-coding regions such as regulatory regions.

As used herein, the terms “coding region” and “coding sequence” are usedinterchangeably and refer to a nucleotide sequence that encodes apolypeptide and, when placed under the control of appropriate regulatorysequences expresses the encoded polypeptide. The boundaries of a codingregion are generally determined by a translation start codon at its 5′end and a translation stop codon at its 3′ end. A “regulatory sequence”is a nucleotide sequence that regulates expression of a coding sequenceto which it is operably linked. Non-limiting examples of regulatorysequences include promoters, enhancers, transcription initiation sites,translation start sites, translation stop sites, and transcriptionterminators. The term “operably linked” refers to a juxtaposition ofcomponents such that they are in a relationship permitting them tofunction in their intended manner. A regulatory sequence is “operablylinked” to a coding region when it is joined in such a way thatexpression of the coding region is achieved under conditions compatiblewith the regulatory sequence.

A polynucleotide that includes a coding region may include heterologousnucleotides that flank one or both sides of the coding region. As usedherein, “heterologous nucleotides” refer to nucleotides that are notnormally present flanking a coding region that is present in a wild-typecell. For instance, a coding region present in a wild-type microbe andencoding a PelA polypeptide is flanked by homologous sequences, and anyother nucleotide sequence flanking the coding region is considered to beheterologous. Examples of heterologous nucleotides include, but are notlimited to regulatory sequences. Typically, heterologous nucleotides arepresent in a polynucleotide of the present invention through the use ofstandard genetic and/or recombinant methodologies well known to oneskilled in the art. A polynucleotide of the present invention may beincluded in a suitable vector.

As used herein, an “exogenous polynucleotide” refers to a polynucleotidethat is not normally or naturally found in a microbe. As used herein,the term “endogenous polynucleotide” refers to a polynucleotide that isnormally or naturally found in a cell microbe. An “endogenouspolynucleotide” is also referred to as a “native polynucleotide.”

The terms “complement” and “complementary” as used herein, refer to theability of two single stranded polynucleotides to base pair with eachother, where an adenine on one strand of a polynucleotide will base pairto a thymine or uracil on a strand of a second polynucleotide and acytosine on one strand of a polynucleotide will base pair to a guanineon a strand of a second polynucleotide. Two polynucleotides arecomplementary to each other when a nucleotide sequence in onepolynucleotide can base pair with a nucleotide sequence in a secondpolynucleotide. For instance, 5′-ATGC and 5′-GCAT are complementary. Theterm “substantial complement” and cognates thereof as used herein, referto a polynucleotide that is capable of selectively hybridizing to aspecified polynucleotide under stringent hybridization conditions.Stringent hybridization can take place under a number of pH, salt andtemperature conditions. The pH can vary from 6 to 9, preferably 6.8 to8.5. The salt concentration can vary from 0.15 M sodium to 0.9 M sodium,and other cations can be used as long as the ionic strength isequivalent to that specified for sodium. The temperature of thehybridization reaction can vary from 30° C. to 80° C., preferably from45° C. to 70° C. Additionally, other compounds can be added to ahybridization reaction to promote specific hybridization at lowertemperatures, such as at or approaching room temperature. Among thecompounds contemplated for lowering the temperature requirements isformamide. Thus, a polynucleotide is typically substantiallycomplementary to a second polynucleotide if hybridization occurs betweenthe polynucleotide and the second polynucleotide. As used herein,“specific hybridization” refers to hybridization between twopolynucleotides under stringent hybridization conditions.

As used herein, the term “polypeptide” refers broadly to a polymer oftwo or more amino acids joined together by peptide bonds. The term“polypeptide” also includes molecules which contain more than onepolypeptide joined by a disulfide bond, or complexes of polypeptidesthat are joined together, covalently or noncovalently, as multimers(e.g., dimers, tetramers). Thus, the terms peptide, oligopeptide,enzyme, and protein are all included within the definition ofpolypeptide and these terms are used interchangeably. It should beunderstood that these terms do not connote a specific length of apolymer of amino acids, nor are they intended to imply or distinguishwhether the polypeptide is produced using recombinant techniques,chemical or enzymatic synthesis, or is naturally occurring.

As used herein, a polypeptide “fragment” includes any polypeptide whichretains at least some of the activity of the corresponding nativepolypeptide. Examples of fragments of polypeptides described hereininclude, but are not limited to, proteolytic fragments and deletionfragments.

As used herein, an “enriched” polypeptide or polynucleotide is one thatconstitutes a significantly higher fraction (2 to 5 fold) of the totalof amino acids or nucleotides present in the cells of interest than inthe cells from which the sequence was separated. A skilled person canpreferentially reduce the amount of other amino acids or nucleotidespresent, or preferentially increase the amount of specific amino acidsequences or nucleotide sequences of interest, or both. However, theterm “enriched” does not imply that there are no other polypeptides orpolynucleotides present. Enriched simply means the relative amount ofthe sequence of interest has been significantly increased. The term“significant” indicates that the level of increase is useful to theperson making such an increase.

As used herein, an “isolated” polypeptide or polynucleotide is one thathas been removed from its natural environment, produced usingrecombinant techniques, or chemically or enzymatically synthesized. Forinstance, a polypeptide or a polynucleotide can be isolated. As usedherein, a purified” substance is one that is at least 80% free,preferably at least 90% free, and most preferably at least 95% free fromother components with which they are naturally associated.

As used herein, “pectinase activity” refers to the ability of apolypeptide to catalyze the depolymerization of the polygalacturonatebackbone of pectin by β-elimination giving a Δ4,5-unsaturated product(Jayani et al., 2005, Process Biochem., 40:2931-2944, Sakai et al.,1993, Adv. Appl. Microbiol., 39:231-294). The polypeptide havingpectinase activity may have pectate lyase activity, pectin lyaseactivity, or both pectate lyase activity and pectin lyase activity.“Pectate lyase activity” refers to the ability of a polypeptide todegrade unesterified polygalacturonate, and “pectin lyase activity”refers to the ability of a polypeptide to degrade methyl esterifiedpectin. A pectinase disclosed herein may degrade pectin tooligogalacturonides having a degree of polymerization of less than 8,less than 7, less than 6, less than 5, less than 4, or less than 3.

As used herein, “oligogalacturonate activity” refers to the ability of apolypeptide to catalyze the degradation of short oligogalacturonates,such as oligogalacturonates with a degree of polymerization less thanseven, to result in dimeric or monomeric sugars.

As used herein, “degrade” and “degradation” refers to the breakdown of apolysaccharide, typically by cleaving a polysaccharide between twosaccharides. A single saccharide may be released if it is at the end ofa polysaccharide, or two shorter polysaccharides may result if thecleavage site is present elsewhere in the polysaccharide. For instance,when the polysaccharide is pectin, a single galacturonic acid may bereleased, or a rhamnose-galacturonic acid disaccharide may be released

As used herein, “identity” refers to sequence similarity between twopolypeptides or two polynucleotides. The sequence similarity between twopolypeptides is determined by aligning the residues of the twopolypeptides (e.g., a candidate amino acid sequence and a referenceamino acid sequence, such as SEQ ID NO:2 or SEQ ID NO:4) to optimize thenumber of identical amino acids along the lengths of their sequences;gaps in either or both sequences are permitted in making the alignmentin order to optimize the number of shared amino acids, although theamino acids in each sequence must nonetheless remain in their properorder. The sequence similarity is typically at least 80% identity, atleast 81% identity, at least 82% identity, at least 83% identity, atleast 84% identity, at least 85% identity, at least 86% identity, atleast 87% identity, at least 88% identity, at least 89% identity, atleast 90% identity, at least 91% identity, at least 92% identity, atleast 93% identity, at least 94% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity, or atleast 99% identity. Sequence similarity may be determined, for example,using sequence techniques such as the BESTFIT algorithm in the GCGpackage (Madison Wis.), or the Blastp program of the BLAST searchalgorithm, available through the World Wide Web, for instance at theinternet site maintained by the National Center for BiotechnologyInformation, National Institutes of Health. Preferably, sequencesimilarity between two amino acid sequences is determined using theBlastp program of the BLAST search algorithm. Preferably, the defaultvalues for all Blastp search parameters are used. In the comparison oftwo amino acid sequences using the Blastp search algorithm, structuralsimilarity is referred to as “identities.”

The sequence similarity between two polynucleotides is determined byaligning the residues of the two polynucleotides (e.g., a candidatenucleotide sequence and a reference nucleotide sequence, such as SEQ IDNO:1 or SEQ ID NO:3) to optimize the number of identical nucleotidesalong the lengths of their sequences; gaps in either or both sequencesare permitted in making the alignment in order to optimize the number ofshared nucleotides, although the nucleotides in each sequence mustnonetheless remain in their proper order. The sequence similarity istypically at least 80% identity, at least 81% identity, at least 82%identity, at least 83% identity, at least 84% identity, at least 85%identity, at least 86% identity, at least 87% identity, at least 88%identity, at least 89% identity, at least 90% identity, at least 91%identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97%identity, at least 98% identity, or at least 99% identity. Sequencesimilarity may be determined, for example, using sequence techniquessuch as GCG FastA (Genetics Computer Group, Madison, Wis.), MacVector4.5 (Kodak/IBI software package) or other suitable sequencing programsor methods known in the art. Preferably, sequence similarity between twonucleotide sequences is determined using the Blastn program of the BLASTsearch algorithm, available through the World Wide Web, for instance atthe internet site maintained by the National Center for BiotechnologyInformation, National Institutes of Health. Preferably, the defaultvalues for all Blastn search parameters are used. In the comparison oftwo nucleotide sequences using the Blastn search algorithm, sequencesimilarity is referred to as “identities.”

Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as an enzymatic reaction, or“suitable” conditions are conditions that do not prevent such eventsfrom occurring. Thus, these conditions permit, enhance, facilitate,and/or are conducive to the event. Such conditions, known in the art anddescribed herein, may depend upon, for example, the enzyme being used.

As used herein, a “microbe” refers to a prokaryotic cell, includingbacteria and archaea, and a eukaryotic cell, including fungi (such asyeast).

As used herein, “genetically modified microbe” refers to a microbe intowhich has been introduced an exogenous polynucleotide, e.g., anexpression vector. For example, a microbe is a genetically modifiedmicrobe by virtue of introduction into a suitable microbe of anexogenous polynucleotide that is foreign to the microbe. “Geneticallymodified microbe” also refers to a microbe that has been geneticallymanipulated such that endogenous nucleotides have been altered. Forexample, a microbe is a genetically modified microbe by virtue ofintroduction into a suitable microbe of an alteration of endogenousnucleotides. For instance, an endogenous coding region could be deletedor mutagenized. Such mutations may result in a polypeptide having adifferent amino acid sequence than was encoded by the endogenouspolynucleotide. Another example of a genetically modified microbe is onehaving an altered regulatory sequence, such as a promoter, to result inincreased or decreased expression of an operably linked endogenouscoding region.

“Metabolic product” refers to any product (e.g., oxalic acid, succinicacid, lactic acid, pyruvic acid, salts thereof, amino acids, ethanol,etc.) from the fermentation of plant biomass, e.g., lignocelluosicbiomass. Metabolic products include, but are not limited to, commoditychemicals such as small organic (e.g., C1-C8) acids such as, forexample, succinic acid, lactic acid, citric acid, oxaloacetic acid,malic acid, adipic acid, fumaric acid, or pyruvic acid, and alcoholssuch as, for example, ethanol, n-butanol, 1,4-butanediol, sec-butanol,and/or methanol.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Integration of casAcasB into the lac operon of E. coli KO11.

FIG. 2. (A) Ethanol production and reducing sugars from sugar beet pulpfermentation for E. coli KO11, LY40A, and JP07C (standard error, n=3;solid lines indicate ethanol concentration and dashed lines representreducing sugar concentrations). (B) Absorbance at 235 nm ofoligogalacturonides with dp≦6 from the above sugar beet pulpfermentation (data represents average of two experiments);  K011; ▾LY40A; ▪ JP07C.

FIG. 3. Ethanol production and reducing sugars from sugar beet pulpfermentation for E. coli KO11, LY40A, JP07C, and JP08C (standard error,n=3; solid lines indicate ethanol concentration and dashed linesrepresent reducing sugar concentrations);  K011; ▾ LY40A; ▪ JP07C; ♦JP08C.

FIG. 4. Amino acid alignment of pectate lyase class 3 enzymes (CLUSTALW). Numbering begins at the N-termini of the proteins. Gaps areindicated by dashes. In the final line, identical amino acids areindicated by asterisks and conserved and semi-conserved residues bycolons and dots, respectively. Family PL3 conserved residues are boxed.Amino acids identical in at least six of the sequences aligned areshaded. BliYvpA, B. licheniformis protein from gene yvpA; BsuPelC, B.subtilis pectate lyase C; BspP2850, Bacillus sp. P-2850 pectate lyase;PamPelA, P. amylolyticus pectate lyase A; PbaPelA, P. barcinonensispectate lyase A; BspKSM15, Bacillus sp. KSM-P15 pectate lyase; EcaPel3,E. carotovora pectate lyase 3; EchPelI, E. chrysanthemi pectate lyase I;and FsoPelB, F. solani pectate lyase B. The conserved arginine, whichmay play a role in the active site, is in bold.

FIG. 5. Amino acid alignment of pectate lyase class 1 enzymes (CLUSTALW). Numbering begins at the N-termini of the proteins. Gaps areindicated by dashes. In the final line, identical amino acids areindicated by asterisks and conserved and semi-conserved residues bycolons and dots, respectively. Pectate lyase conserved sequence patternsare boxed, vWIDH, AxDIKGxxxxVTxS, and VxxRxPxxRxGxxHxxxxN (Henrissat etal., 1995, Plant Physiol., 107:963-976). Residues of sites conserved inall thermostable PL1 pectate lyase are highlighted in grey, conservedcatalytic sites are highlighted in black, and conserved calcium bindingsites are labeled with © symbol. TmaPelA, T. maritime MSB8 pectate lyaseA; BsuBS2, B. subtilis BS-2 pectate lyase; BamPel, B. amyloliquefaciensTB-2 pectate lyase; BspYA14, Bacillus sp. YA-14 pectate lyase K; BsuPel,B. subtilis reference strain 168 pectate lyase; BliPel, B. licheniformisATCC 14580 pectate lyase; and PamPelB, P. amylolyticus pectate lyase B.

FIG. 6. PelA optima for pH (A), temperature (B), and CaCl₂ (C).

FIG. 7. Activity on different pectic substrates for P. amylolyticuspectate lyase A, PamPelA; P. barcinonensis pectate lyase A, PbaPelA; andB. subtilis pectate lyase C, BsuPelC.

FIG. 8. PelB optima for pH (A), temperature (B), and CaCl₂ (C).

FIG. 9. P. amylolyticus pectate lyase B activity on different pecticsubstrates.

FIG. 10. Comparison of oligogalacturonides with a dp<7 after growth onsugar beet pulp for E. coli DH5α with pUC19 () or p13C2 (▾).

FIG. 11. Ethanol production and reducing sugars from sugar beet pulpfermentation for E. coli LY40A () and JP27 (▾) (standard error, n=3;solid lines indicate ethanol concentration and dashed line representreducing sugar concentrations).

FIG. 12. Nucleotide sequence (SEQ ID NO:1) encoding a PelA polypeptide(SEQ ID NO:2), nucleotide sequence (SEQ ID NO:3) encoding a PelBpolypeptide (SEQ ID NO:4), and nucleotide sequence (SEQ ID NO:5)encoding an Ogl polypeptide (SEQ ID NO:6).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes isolated polypeptides having pectinaseactivity. One type of polypeptide having pectinase activity is referredto herein as a PelA polypeptide. An example of a PelA polypeptide isdepicted at SEQ ID NO:2. Other examples of PelA polypeptides of thepresent invention include those having sequence similarity with theamino acid sequence of SEQ ID NO:2. A PelA polypeptide having sequencesimilarity with the amino acid sequence of SEQ ID NO:2 has pectinaseactivity. A PelA polypeptide may be isolated from a microbe, such as amember of the genera Paenibacillus, preferably P. amylolyticus, or maybe produced using recombinant techniques, or chemically or enzymaticallysynthesized using routine methods.

The amino acid sequence of a PelA polypeptide having sequence similarityto SEQ ID NO:2 may include conservative substitutions of amino acidspresent in SEQ ID NO:2. A conservative substitution is typically thesubstitution of one amino acid for another that is a member of the sameclass. For example, it is well known in the art of protein biochemistrythat an amino acid belonging to a grouping of amino acids having aparticular size or characteristic (such as charge, hydrophobicity,and/or hydrophilicity) may generally be substituted for another aminoacid without substantially altering the secondary and/or tertiarystructure of a polypeptide. Conservative amino acid substitutions canresult from exchange of amino acids residues from within one of thefollowing classes of residues: Class I: Gly, Ala, Val, Leu, and Ile(representing aliphatic side chains); Class II: Gly, Ala, Val, Leu, Ile,Ser, and Thr (representing aliphatic and aliphatic hydroxyl sidechains); Class III: Tyr, Ser, and Thr (representing hydroxyl sidechains); Class IV: Cys and Met (representing sulfur-containing sidechains); Class V: Glu, Asp, Asn and Gln (carboxyl or amide groupcontaining side chains); Class VI: His, Arg and Lys (representing basicside chains); Class VII: Gly, Ala, Pro, Trp, Tyr, Ile, Val, Leu, Phe andMet (representing hydrophobic side chains); Class VIII: Phe, Trp, andTyr (representing aromatic side chains); and Class IX: Asn and Gln(representing amide side chains). The classes are not limited tonaturally occurring amino acids, but also include artificial aminoacids, such as beta or gamma amino acids and those containingnon-natural side chains, and/or other similar monomers such ashydroxyacids. A portion of SEQ ID NO:2 is shown in FIG. 4 in a multipleprotein alignment with other pectate lyase class 3 enzymes. Identicalamino acids are marked with an asterisk, and conserved andsemi-conserved amino acids are marked with colons and dots,respectively.

Guidance concerning how to make phenotypically silent amino acidsubstitutions is provided in Bowie et al. (1990, Science,247:1306-1310), wherein the authors indicate proteins are surprisinglytolerant of amino acid substitutions. For example, Bowie et al. disclosethat there are two main approaches for studying the tolerance of apolypeptide sequence to change. The first method relies on the processof evolution, in which mutations are either accepted or rejected bynatural selection. The second approach uses genetic engineering tointroduce amino acid changes at specific positions of a cloned gene andselects or screens to identify sequences that maintain functionality. Asstated by the authors, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which changes are likely to be permissive at a certain positionof the protein. For example, most buried amino acid residues requirenon-polar side chains, whereas few features of surface side chains aregenerally conserved. Other such phenotypically silent substitutions aredescribed in Bowie et al, and the references cited therein.

The present invention also includes isolated polynucleotides encoding apolypeptide of the present invention, e.g., a PelA polypeptide. Apolynucleotide encoding a PelA polypeptide is referred to herein as aPelA polynucleotide. PelA polynucleotides may have a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:2. An example of the class of nucleotide sequences encoding such apolypeptide is SEQ ID NO:1. It should be understood that apolynucleotide encoding an PelA polypeptide represented by SEQ ID NO:2is not limited to the nucleotide sequence disclosed at SEQ ID NO:1, butalso includes the class of polynucleotides encoding such polypeptides asa result of the degeneracy of the genetic code. For example, thenaturally occurring nucleotide sequence SEQ ID NO:1 is but one member ofthe class of nucleotide sequences encoding a polypeptide having theamino acid sequence SEQ ID NO:2. The class of nucleotide sequencesencoding a selected polypeptide sequence is large but finite, and thenucleotide sequence of each member of the class may be readilydetermined by one skilled in the art by reference to the standardgenetic code, wherein different nucleotide triplets (codons) are knownto encode the same amino acid.

A PelA polynucleotide of the present invention may have sequencesimilarity with the nucleotide sequence of SEQ ID NO:1. PelApolynucleotides having sequence similarity with the nucleotide sequenceof SEQ ID NO:1 encode a PelA polypeptide. A PelA polynucleotide may beisolated from a microbe, such as a member of the genera Paenibacillus,preferably P. amylolyticus, or may be produced using recombinanttechniques, or chemically or enzymatically synthesized. A PelApolynucleotide of the present invention may further include heterologousnucleotides flanking the open reading frame encoding the PelApolynucleotide. Typically, heterologous nucleotides may be at the 5′ endof the coding region, at the 3′ end of the coding region, or thecombination thereof. The number of heterologous nucleotides may be, forinstance, at least 10, at least 100, or at least 1000.

Another type of polypeptide having pectinase activity is referred toherein as a PelB polypeptide. An example of a PelB polypeptide isdepicted at SEQ ID NO:4. Other examples of PelB polypeptides of thepresent invention include those having sequence similarity with theamino acid sequence of SEQ ID NO:4. A PelB polypeptide having sequencesimilarity with the amino acid sequence of SEQ ID NO:4 has pectinaseactivity. A PelB polypeptide may be isolated from a microbe, such as amember of the genera Paenibacillus, preferably P. amylolyticus, or maybe produced using recombinant techniques, or chemically or enzymaticallysynthesized using routine methods.

The amino acid sequence of a PelB polypeptide having sequence similarityto SEQ ID NO:4 may include conservative substitutions of amino acidspresent in SEQ ID NO:4. A portion of SEQ ID NO:4 is shown in FIG. 5 in amultiple protein alignment with other pectate lyase class 1 enzymes.Identical amino acids are marked with an asterisk, and conserved andsemi-conserved amino acids are marked with colons and dots,respectively. Conserved regions are boxed, residues of sites conservedin all thermostable PL1 pectate lyases are highlighted in grey,conserved catalytic sites are highlighted in black, and conservedcalcium binding sites are labeled with the © symbol. Furthercharacteristics of PelB polypeptides are disclosed in Example 4.

The present invention also includes isolated polynucleotides encoding apolypeptide of the present invention, e.g., a PelB polypeptide. Apolynucleotide encoding a PelB polypeptide is referred to herein as aPelB polynucleotide. PelB polynucleotides may have a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:4. An example of the class of nucleotide sequences encoding such apolypeptide is SEQ ID NO:3. It should be understood that apolynucleotide encoding a PelB polypeptide represented by SEQ ID NO:4 isnot limited to the nucleotide sequence disclosed at SEQ ID NO:3, butalso includes the class of polynucleotides encoding such polypeptides asa result of the degeneracy of the genetic code. For example, thenaturally occurring nucleotide sequence SEQ ID NO:3 is but one member ofthe class of nucleotide sequences encoding a polypeptide having theamino acid sequence SEQ ID NO:4. The class of nucleotide sequencesencoding a selected polypeptide sequence is large but finite, and thenucleotide sequence of each member of the class may be readilydetermined by one skilled in the art by reference to the standardgenetic code, wherein different nucleotide triplets (codons) are knownto encode the same amino acid.

A PelB polynucleotide of the present invention may have sequencesimilarity with the nucleotide sequence of SEQ ID NO:3. PelBpolynucleotides having sequence similarity with the nucleotide sequenceof SEQ ID NO:3 encode a PelB polypeptide. A PelB polynucleotide may beisolated from a microbe, such as a member of the genera Paenibacillus,preferably P. amylolyticus, or may be produced using recombinanttechniques, or chemically or enzymatically synthesized. A PelBpolynucleotide of the present invention may further include heterologousnucleotides flanking the open reading frame encoding the PelBpolynucleotide. Typically, heterologous nucleotides may be at the 5′ endof the coding region, at the 3′ end of the coding region, or thecombination thereof. The number of heterologous nucleotides may be, forinstance, at least 10, at least 100, or at least 1000.

Whether a polypeptide has pectinase activity may be determined by invitro assays. Preferably, an in vitro assay is carried out essentiallyas described (Collmer et al., 1988, In: Wood & Kellogg (Eds.) Methods inEnzymology. San Diego, Calif., Academic Press, Inc., Soriano et al.,2000, Microbiology, 146:89-95). Briefly, a polypeptide to be tested forpectinase activity may be expressed in a cell, such as a geneticallymodified microbial cell, and a cell extract may be prepared by, forinstance, sonication. A standard enzyme assay mixture may include 0.2%weight/volume (w/v) of the substrate. Suitable substrates includeunesterified polygalacturonic acid or an esterified pectin. Examples ofsuitable esterified pectins include those having between 8.5% and 90%esterification. The substrate may be present in a final volume of 1 mLof 50 mM glycine buffer containing CaCl₂. The pH of the buffer may bebetween 9 and 10.5; however, when the polypeptide has sequencesimilarity to a PelA polypeptide the pH may be between 10.25 and 10.75,such as 10.5, and when the polypeptide has sequence similarity to a PelBpolypeptide the pH may be between 9.25 and 9.75, such as 9.5. The CaCl₂concentration may be between 0.3 mM and 1.75 mM; however, when thepolypeptide has sequence similarity to a PelA polypeptide the CaCl₂concentration may be between 1.25 mM and 1.75 mM, such as 1.5 mM, andwhen the polypeptide has sequence similarity to a PelB polypeptide theCaCl₂ concentration may be between 0.3 mM and 0.7 mM, such as 0.5 mM.The assay mixture and enzyme preparation may be equilibrated to anappropriate temperature and monitored for the formation ofΔ-4,5-unsaturated products at 235 nm for 1 to 3 min. The temperature ofthe reaction may be between 40° C. and 57° C.; however, when thepolypeptide has sequence similarity to a PelA polypeptide thetemperature may be between 42° C. and 47° C., such as 45° C., and whenthe polypeptide has sequence similarity to a PelB polypeptide thetemperature may be between 53° C. and 57° C., such as 55° C. One unit ofenzyme activity is defined as the amount of enzyme that produces 1 mmol4,5-unsaturated product per minute.

The present invention also includes fragments of the polypeptidesdescribed herein, and the polynucleotides encoding such fragments, PelApolypeptides and PelB polypeptides, such as SEQ ID NOs:2 and 4,respectively. A polypeptide fragment may include a sequence of at least5, at least 10, at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, at least 50, at least 55, at least60, at least 65, at least 70, at least 75, at least 80, at least 85, atleast 90, at least 95, or at least 100 amino acid residues.

A polypeptide of the present invention or a fragment thereof may beexpressed as a fusion polypeptide that includes a polypeptide of thepresent invention or a fragment thereof and an additional amino acidsequence. For instance, the additional amino acid sequence may be usefulfor purification of the fusion polypeptide by affinity chromatography.Various methods are available for the addition of such affinitypurification moieties to proteins. Representative examples may be foundin Hopp et al. (U.S. Pat. No. 4,703,004), Hopp et al. (U.S. Pat. No.4,782,137), Sgarlato (U.S. Pat. No. 5,935,824), and Sharma (U.S. Pat.No. 5,594,115). In another example, the additional amino acid sequencemay be a carrier polypeptide. The carrier polypeptide may be used toincrease the immunogenicity of the fusion polypeptide to increaseproduction of antibodies that specifically bind to a polypeptide of theinvention. The invention is not limited by the types of carrierpolypeptides that may be used to create fusion polypeptides. Examples ofcarrier polypeptides include, but are not limited to, keyhole limpethemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbitserum albumin, and the like.

Another example of an additional amino acid sequence is a secretorysequence. In certain embodiments, for instance, where a polynucleotideencoding a PelA polypeptide of the present invention or a fragmentthereof, a PelB polypeptide or a fragment thereof, or a combinationthereof is expressed in a prokaryotic cell, the polypeptide may includea signal sequence that is present at the amino terminal end. The signalsequence targets the polypeptide for export out of the cytoplasm of thecell. Signal sequences that function in eukaryotic cells and inprokaryotic cells are known to the skilled person and are used routinelyto engineer polypeptides for export.

A polynucleotide of the present invention may be present in a vector. Avector is a replicating polynucleotide, such as a plasmid, phage, orcosmid, to which another polynucleotide may be attached so as to bringabout the replication of the attached polynucleotide. Construction ofvectors containing a polynucleotide of the invention employs standardligation techniques known in the art. See, e.g., Sambrook et al,Molecular Cloning: A Laboratory Manual., Cold Spring Harbor LaboratoryPress (1989). A vector may provide for further cloning (amplification ofthe polynucleotide), i.e., a cloning vector, or for expression of thepolynucleotide, i.e., an expression vector. The term vector includes,but is not limited to, plasmid vectors, viral vectors, cosmid vectors,and artificial chromosome vectors. Examples of viral vectors include,for instance, lambda phage vectors, P1 phage vectors, M13 phage vectors,adenoviral vectors, adeno-associated viral vectors, lentiviral vectors,retroviral vectors, and herpes virus vectors. Typically, a vector iscapable of replication in a microbial host, for instance, a fungus, suchas S. cerevisiae, or a prokaryotic bacterium, such as E. coli.Preferably the vector is a plasmid.

Selection of a vector depends upon a variety of desired characteristicsin the resulting construct, such as a selection marker, vectorreplication rate, and the like. In some aspects, suitable host cells forcloning or expressing the vectors herein include eukaryotic cells.Suitable eukaryotic cells include fungi, such as S. cerevisiae and P.pastoris. In other aspects, suitable host cells for cloning orexpressing the vectors herein include prokaryotic cells. Suitableprokaryotic cells include eubacteria, such as gram-negative microbes,for example, E. coli. Vectors may be introduced into a host cell usingmethods that are known and used routinely by the skilled person. Forexample, calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acids into host cells.

Polynucleotides of the present invention may be obtained from microbes,for instance, members of the genus Paenibacillus, such as P.amylolyticus. Members of the genus Paenibacillus useful in the methodsdisclosed herein may be obtained from soil, such as soil containingorganic material, for example rice fields, food products (Yoshikatsu etal., 2006, Biocontro. Sci., 11:43-47; Kim et al., 2009, Int. J. Syst.Evol. Microbiol., 59:1002-1006), or the digestive tract of insects thathave a diet that includes lignocellulosic biomass, for instance,termites, honeybee (Neuendorf et al., 2004, Microbiol., 150:2381-2390),and Tipula abdominalis (Cook et al., 2007, Appl. Environ. Microbiol.,73:5683-5686). Polynucleotides of the present invention may be producedin vitro or in vivo. For instance, methods for in vitro synthesisinclude, but are not limited to, chemical synthesis with a conventionalDNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotidesand reagents for such synthesis are well known. Likewise, polypeptidesof the present invention may be obtained from microbes, or produced invitro or in vivo.

An expression vector optionally includes regulatory sequences operablylinked to the coding region. The invention is not limited by the use ofany particular promoter, and a wide variety of promoters are known.Promoters act as regulatory signals that bind RNA polymerase in a cellto initiate transcription of a downstream (3′ direction) coding region.The promoter used may be a constitutive or an inducible promoter. It maybe, but need not be, heterologous with respect to the host cell.Examples of promoters include, but are not limited to, promoters thatfunction in anaerobic conditions and promoters that are not subject toinhibition by glucose.

An expression vector may optionally include a ribosome binding site anda start site (e.g., the codon ATG) to initiate translation of thetranscribed message to produce the polypeptide. It may also include atermination sequence to end translation. A termination sequence istypically a codon for which there exists no correspondingaminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotideused to transform the host cell may optionally further include atranscription termination sequence.

A vector introduced into a host cell optionally includes one or moremarker sequences, which typically encode a molecule that inactivates orotherwise detects or is detected by a compound in the growth medium. Forexample, the inclusion of a marker sequence may render the transformedcell resistant to an antibiotic, or it may confer compound-specificmetabolism on the transformed cell. Examples of a marker sequence aresequences that confer resistance to kanamycin, ampicillin,chloramphenicol, tetracycline, and neomycin.

The present invention also includes antibodies that specifically bind apolypeptide of the present invention. An antibody that specificallybinds a PelA polypeptide of the present invention, preferably, SEQ IDNO:2 or a fragment thereof, does not bind to a pectate lyase expressedby P. barcinonensis, and described at Genbank accession number CAB40884.An antibody that specifically binds a PelB polypeptide of the presentinvention, preferably, SEQ ID NO:4 or a fragment thereof, does not bindto a pectate lyase expressed by Bacillus sp. YA-14, and described atGenbank accession number BAA05383.

Antibody may be produced using a polypeptide of the present invention,or a fragment thereof. The antibody may be polyclonal or monoclonal.Laboratory methods for producing, characterizing, and optionallyisolating polyclonal and monoclonal antibodies are known in the art(see, for instance, Harlow E. et al., 1988, Antibodies: A laboratorymanual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Forinstance, a polypeptide of the present invention may be administered toan animal, preferably a mammal, in an amount effective to cause theproduction of antibody specific for the administered polypeptide.Optionally, a polypeptide may be mixed with an adjuvant, for instanceFreund's incomplete adjuvant, to stimulate the production of antibodiesupon administration. Whether an antibody of the present inventionspecifically binds to a polypeptide of the present invention may bedetermined using methods known in the art. For instance, specificity maybe determined by testing antibody binding to SEQ ID NO:2 and apolypeptide having the amino acid sequence described at Genbankaccession number CAB40884. Other examples include testing the kineticsof antibody binding to different polypeptides, and testing competitionin binding using as competitors known polypeptides containing or notcontaining an epitope against which the antibody is directed.

The present invention also includes genetically modified microbes andcompositions that include genetically modified microbes. In someembodiments a genetically modified microbe has a polynucleotide encodinga polypeptide having pectinase activity, such as a PelA polypeptide, aPelB polypeptide, or a combination thereof. Compared to a controlmicrobe that is not genetically modified, a genetically modified microbemay exhibit production of a PelA polypeptide or a fragment thereof,production of a PelB polypeptide or a fragment thereof, or thecombination thereof. A polynucleotide encoding a PelA polypeptide, aPelB polypeptide, or a combination thereof, may be present in themicrobe as a vector or integrated into a chromosome.

Examples of microbes that can be genetically modified to encode apolypeptide having pectinase activity include, but are not limited to,microbes known to be capable of producing cellulolytic enzymes, e.g.,species of Humicola, Coprinus, Thielavia, Fusarium, Myceliophthora,Acremonium, Cephalosporium, Scytalidium, Penicillium or Aspergillus(see, for example, Shun-Ichi et al., U.S. Pat. No. 5,258,297),especially those produced by a strain selected from the species Humicolainsolens (reclassified as Scytalidium thermophilum, see for example,Barbesgaard et al., U.S. Pat. No. 4,435,307), Coprinus cinereus,Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus,Thielavia terrestris, Acremonium sp., Acremonium persicinum, Acremoniumacremonium, Acremonium brachypenium, Acremonium dichromosporum,Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum,Acremonium incoloratum, and Acremonium furatum; preferably from thespecies Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672,Myceliophthora thermophila CBS 117.65, Cephalosporium sp. RYM-202,Acremonium sp. CBS 478.94, Acremonium sp. CBS 265.95, Acremoniumpersicinum CBS 169.65, Acremonium acremonium AHU 9519, Cephalosporiumsp. CBS 535.71, Acremonium brachypenium CBS 866.73, Acremoniumdichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74, Acremoniumpinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56, Acremoniumincoloratum CBS 146.62, and Acremonium furatum CBS 299.70H. Cellulolyticenzymes may also be obtained from Trichoderma (particularly Trichodermaviride, Trichoderma reesei, and Trichoderma koningii), alkalophilicBacillus (see, for example, Horikoshi et al., U.S. Pat. No. 3,844,890and Shun-Ichi et al., U.S. Pat. No. 5,258,297), and Streptomyces (see,for example, Shun-Ichi et al., U.S. Pat. No. 5,258,297).

Examples of microbes that can be genetically modified to encode apolypeptide having pectinase activity include, but are not limited to,microbes known to be capable of producing ethanol. Useful eukaryoticcells include, but are not limited to, Saccharomyces (such asSaccharomyces cerevisiae), and Pichia (such as Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichiamethanolica).

Examples of S. cerevisiae include, but are not limited to, Baker'syeast, Tembec T1 (Keating et al., 2004, J. Ind. Microbiol. Biotechnol.31:235), Y-1528 (Keating et al., 2004, J. Ind. Microbiol. Biotechnol.31:235), TMB3000 (Alkasrawi et al., 2006, Enzyme Microb. Tech., 38:279),CBS 8066, CEN/PK 113-7D, TMB3500, USM21, and NRRLY-12632. Examples ofPichia stipitis include, but are not limited to, NRRLY-7124. Otherexamples of commercially available yeast which can be used include, forinstance, RED STAR and ETHANOL RED yeast (available fromFermentis/Lesaffre, USA), FALI (available from Fleischmann's Yeast,USA), SUPERSTART and THERMOSACC fresh yeast (available from EthanolTechnology, Wis., USA), BIOFERM AFT and XR (available from NABC—NorthAmerican Bioproducts Corporation, GA, USA), GERT STRAND (available fromGert Strand AB, Sweden), and FERMIOL (available from DSM Specialties).

Examples of prokaryotic ethanologenic microbes include, but are notlimited to, Escherichia, in particular Escherichia coli, members of thegenus Zymomonas, in particular Zymomonas mobilis, members of the genusZymobacter, in particular Zymobactor palmae, members of the genusKlebsiella, in particular Klebsiella oxytoca, members of the genusLeuconostoc, in particular Leuconostoc mesenteroides, members of thegenus Lactobacillus, in particular Lactobacillus helveticus andLactobacillus delbruckii, members of the genus Lactococcus, inparticular Lactococcus lactis, members of the genus Clostridium, inparticular Clostridium butyricum, members of the genus Enterobacter, inparticular Enterobacter aerogenes, and members of the genusThermoanaerobacter, in particular Thermoanaerobacter BG1L1,Thermoanarobacter ethanolicus, Thermoanaerobacter thermosaccharolyticum,or Thermoanaerobacter mathranii. Examples of E. coli include, but arenot limited to, KO11, LY01.

A genetically modified microbe of the present invention may includeother modifications. For instance, a genetically modified microbe of thepresent invention may include other modifications that provide forincreased ability to use renewable resources, such as lignocellulosicbiomass, for the production of desired metabolic products, such ascommodity chemicals. Modifications may provide for increased productionof commodity chemicals by, for instance, increasing production ofenzymes in metabolic pathways, reducing feedback inhibition at differentlocations in metabolic pathways, increasing importation of substratesused in metabolic pathways to produce a commodity chemical, and/orincreasing secretion of polypeptides. Polypeptides involved in thedegradation of polysaccharides to glucose, xylose, mannose, galactose,and arabinose include, for instance, endoglucanases, cellobiohydrolases,glucohydrolases, beta-glucosidases, methylesterases, depolymerases,pectin and pectate lyases, and cellobioases (e.g., casAB coding regions,such as those described at Genbank accession U61727).

Polypeptides involved in producing metabolic products, such as commoditychemicals, can vary with the chemical being produced, and may includethose useful when the substrate includes a 6-carbon sugar and/or a5-carbon sugar. For instance, when lactic acid is to be produced thegenetically modified microbe may include polynucleotides encoding alactate dehydrogenase that catalyses the formation of L-(+) or D-(−)lactic acid. When ethanol is to be produced the genetically modifiedmicrobe may include polynucleotides encoding a pyruvate decarboxylase,an alcohol dehydrogenase, and/or a phosphotransferase (Ingram et al.,U.S. Pat. No. 6,102,690, Ingram et al., U.S. Pat. No. 7,026,152). Underanaerobic conditions pyruvate is converted to acetyl CoA, catalysed bythe enzyme pyruvate formate lyase (PFL). Acetyl CoA is subsequentlyconverted into acetaldehyde by the enzyme acetaldehyde dehydrogenase(AcDH) and ethanol is produced by the reduction of acetaldehydecatalyzed by ADH. When butanol is to be produced the geneticallymodified microbe may include polynucleotides encoding a3-hydroxybutyryl-CoAdehydrogen-ase, a crotonase, abutyryl-CoAdehydrogenase, and/or an aldehyde/alcoholdehydrogenase(Atsumi et al., 2008, Metabolic Engineering, 10:305-311). A geneticallymodified microbe may be engineered to include exogenous polynucleotidesencoding useful enzymes, or endogenous polynucleotides may be modified,for instance, to increase expression of an endogenous coding region.Metabolic pathways of microbes are known to the skilled person andmetabolic engineering to modify the production metabolic products isroutinely practiced. Coding regions encoding polypeptides involved inmetabolic pathways are also known to the skilled person and readilyavailable.

In other aspects, modifications can include disrupting the activity ofone or more endogenous coding regions in a way that inhibits theproduction of non-desired metabolic products and/or redirects themetabolism of intermediates toward the production of desired metabolicproducts. Examples of modifications that disrupt a metabolic pathwayinclude, for example, “knock out” mutations that significantly reduce oreliminate biological activity of the mutated coding region (and/or thepolypeptide encoded by the mutated coding region). Methods forintroducing knock out mutations in many cellular models are routine andknown to those skilled in the art. In other words, one may directmetabolism toward pathways that produce desired products by reducing oreliminating metabolism via pathways that compete with the desiredpathway for metabolic resources.

In those embodiments where a genetically modified microbe includes apectinase, such as a PelA polypeptide or a fragment thereof, or a PelBpolypeptide or a fragment thereof, the genetically modified microbe mayalso include a polynucleotide encoding an enzyme havingoligogalacturonate activity. One type of polypeptide havingoligogalacturonate activity is referred to herein as anoligogalacturonide lyase (Ogl) polypeptide (Reverchon et al., 1989,Gene, 85:125-134, Shevchik et al., 1999, J. Bacteriol., 181:3912-3919).Ogl polypeptides are known to the art, and an example of anoligogalacturonide lyase is described at Genbank Accession numberAAA24825 (SEQ ID NO:6). Other examples of Ogl polypeptides of thepresent invention include those having sequence similarity with theamino acid sequence of SEQ ID NO:6. An Ogl polypeptide having sequencesimilarity with the amino acid sequence of SEQ ID NO:6 hasoligogalacturonate activity. The amino acid sequence of an Oglpolypeptide having sequence similarity to SEQ ID NO:6 may includeconservative substitutions of amino acids present in SEQ ID NO:6.Methods for detecting and measuring oligogalacturonate activity usingdirect UV detection are known to the skilled person and routinely used(Shevchik et al., 1999, J. Bacteriol., 181:3912-3919).

A polynucleotide encoding an Ogl polypeptide is referred to herein as anOgl polynucleotide. Ogl polynucleotides may have a nucleotide sequenceencoding a polypeptide having the amino acid sequence shown in SEQ IDNO:6. An example of the class of nucleotide sequences encoding such apolypeptide is SEQ ID NO:5. It should be understood that apolynucleotide encoding an Ogl polypeptide represented by SEQ ID NO:6 isnot limited to the nucleotide sequence disclosed at SEQ ID NO:5, butalso includes the class of polynucleotides encoding such polypeptides asa result of the degeneracy of the genetic code. For example, thenaturally occurring nucleotide sequence SEQ ID NO:5 is but one member ofthe class of nucleotide sequences encoding a polypeptide having theamino acid sequence SEQ ID NO:6.

An Ogl polynucleotide of the present invention may have sequencesimilarity with the nucleotide sequence of SEQ ID NO:5. Oglpolynucleotides having sequence similarity with the nucleotide sequenceof SEQ ID NO:5 encode an Ogl polypeptide. An Ogl polynucleotide may beisolated from a microbe, such as a member of the genera Erwinia,preferably Erwinia chrysanthemi, or may be produced using recombinanttechniques, or chemically or enzymatically synthesized. An Oglpolynucleotide may further include heterologous nucleotides flanking theopen reading frame encoding the Ogl polynucleotide.

A genetically modified microbe of the present invention may includeother modifications that provide for export of a polypeptide from thecytoplasm of a cell to the exterior of the cell. In some embodiments,such modifications include the addition of polynucleotides encodingpolypeptides that act to impart secretory activity to a gram-negativecell. Examples of secretory systems in gram-negative microbes includeType I, Type II, Type III, Type IV, and the Type VI secretion systems.Examples of useful secretory systems include, but are not limited to,the out system present in Erwinia spp., or the pul system present inKlebsiella spp. (Pugsley et al., 1993, Microbiological Reviews,57:50-108; Lindeberg et al., 1996, Mol. Micro., 20:175-190; Lindeberg etal., 1992, J. Bacteriol., 174:7385-7397; He et al., 1991, Proc. Natl.Acad. Sci. USA, 88:1079-1083). The introduction of one or more secretorypolypeptides into a genetically modified microbe may result in anincrease in the secretion of the selected polypeptide, e.g., apectinase, as compared to secretion of the polypeptide in the cellwithout the secretory polypeptides. The increase in secretion may be atleast 10%, at least 100%, at least 200%, at least 300%, at least 400%,at least 500%, at least 600%, at least 700%, at least 800%, at least900%, or at least 1000%, as compared to levels of secretion in the cellwithout the secretory polypeptides.

Also included in the present invention are methods of making thepolypeptides, polynucleotides, and genetically modified microbesdescribed herein. Polypeptides may be obtained from a microbe thatnaturally produces a polypeptide of the present invention, for instance,a Paenibacillus spp., such as P. amylolyticus. Alternatively, agenetically modified microbe may be used. The methods may includeculturing a microbe under conditions suitable for expression of thepolypeptide, and recovering the polypeptide. The polypeptide may berecovered from the culture medium by conventional procedures includingseparating the cells from the medium by centrifugation or filtration, orif necessary, disrupting the cells and separating the supernatant fromthe cellular fragments and debris. Typically, the proteinaceouscomponents of the supernatant or filtrate are precipitated by means of asalt, e.g., ammonium sulfate. Optionally, the precipitated polypeptidesmay be solubilized and isolated or purified by a variety ofchromatographic procedures, e.g., ion exchange chromatography, affinitychromatography or another similarly art-recognized procedure.Polypeptides and fragments thereof useful in the present invention maybe produced using recombinant DNA techniques, such as an expressionvector present in a cell. Such methods are routine and known in the art.The polypeptides and fragments thereof may also be synthesized in vitro,e.g., by solid phase peptide synthetic methods. The solid phase peptidesynthetic methods are routine and known in the art. A polypeptideproduced using recombinant techniques or by solid phase peptidesynthetic methods may be further purified by routine methods, such asfractionation on immunoaffinity or ion-exchange columns, ethanolprecipitation, reverse phase HPLC, chromatography on silica or on ananion-exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammoniumsulfate precipitation, gel filtration using, for example, Sephadex G-75,or ligand affinity. Also included in the present invention arecompositions that include a PelA polypeptide or a fragment thereof, or aPelB polypeptide or a fragment thereof.

Provided herein are methods for using the polypeptides, polynucleotides,and genetically modified microbes described herein. In one embodiment,the methods include degrading pectin to produce metabolic products.Methods for degrading pectin to produce metabolic products may includeculturing a genetically engineered microbe described herein in acomposition that includes pectin under conditions suitable for degradingthe pectin. Typically, the pectin is present in a lignocellulosicmaterial. Any suitable lignocellulosic material is contemplated incontext of the present methods. Lignocellulosic material may be anymaterial containing lignocellulose and pectin. In some aspects, thelignocellulosic material contains at least 40 wt %, at least 50 wt %, atleast 60 wt %, at least 70 wt %, at least 80 wt %, at least 90 wt %lignocellulose. It is to be understood that the lignocellulosic materialmay also include other constituents such as cellulosic material, such ascellulose, hemicellulose, and may also include constituents such assugars, such as fermentable sugars and/or un-fermentable sugars.

Lignocellulosic material useful in the methods described herein isgenerally found, for example, fruits, such as apple, pear, grape,strawberry, raspberry, blackberry, apricot, mango, guava, papaya,pineapple, and banana, and members of the genus Citrus, such as lemon,lime, orange, tangerine, grapefruit. Sources of lignocellulosicmaterials include other plants such as vegetables, including sugarbeets, soy beans, carrots, tomatoes, and the like. Other examples oflignocellulosic material useful in the methods described herein includeagricultural residues, such as wheat straw, corn stover, pulps such ascitrus pulp and sugar beet pulp, and pomace. It is understood thatlignocellulose material may be in the form of plant cell wall materialcontaining lignin, cellulose, hemicellulose, and pectin in a mixedmatrix.

The pectin may be unesterified, or may be esterified. If the pectin isesterified, the level of esterification may be at least 8.5%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, or at least 80%, no greater than 90%, no greater than 80%, nogreater than 70%, no greater than 60%, no greater than 50%, no greaterthan 40%, or no greater than 30%, or a combination thereof.

A process of producing a metabolic product from lignocellulosicmaterials may include pretreatment, enzymatic hydrolysis through the useof enzymes such as cellulases, fermentation, and/or recovery of themetabolic product. The process may also include, for instance,separation of the sugar solution from residual materials such as lignin.

Biomass from agricultural residues, like sugar beet pulp, may notrequire thermochemical or mechanical pretreatments because they arealready partially processed; however, in certain embodimentspretreatment may be desirable. There are numerous pretreatment methodsor combinations of pretreatment methods known in the art and routinelyused. Physical pretreatment breaks down the size of lignocellulosicmaterial by milling or aqueous/steam processing. Chipping or grindingmay be used to typically produce particles between 0.2 and 30 mm insize. Methods used for lignocelluosic materials typically requireintense physical pretreatments such as steam explosion and other suchtreatments (Peterson et al., U.S. Patent Application 20090093028). Themost common chemical pretreatment methods used for lignocellulosicmaterials include dilute acid, alkaline, organic solvent, ammonia,sulfur dioxide, carbon dioxide or other chemicals to make the biomassmore available to enzymes. Biological pretreatments are sometimes usedin combination with chemical treatments to solubilize the lignin inorder to make cellulose more accessible to hydrolysis and fermentation.

Steam explosion is a common method for pretreatment of lignocellulosicbiomass and increases the amount of cellulose available for enzymatichydrolysis (Foody, U.S. Pat. No. 4,461,648). Generally, the material istreated with high-pressure saturated steam and the pressure is rapidlyreduced, causing the materials to undergo an explosive decompression.Steam explosion is typically initiated at a temperature of 160-260° C.for several seconds to several minutes at pressures of up to 4.5 to 5MPa. The biomass is then exposed to atmospheric pressure. The processtypically causes hemicellulose degradation and lignin transformation.Addition of H₂SO₄, SO₂, or CO₂ to the steam explosion reaction canimprove subsequent cellulose hydrolysis, decrease production ofinhibitory compounds and lead to the more complete removal ofhemicellulose (Morjanoff and Gray, 1987, Biotechnol. Bioeng.29:733-741).

In ammonia fiber explosion (AFEX) pretreatment, biomass is treated withapproximately 1-2 kg ammonia per kg dry biomass for approximately 30minutes at pressures of 1.5 to 2 MPa. (Dale, U.S. Pat. No. 4,600,590;Dale, U.S. Pat. No. 5,037,663; Mes-Hartree, et al. 1988, Appl.Microbiol. Biotechnol., 29:462-468). Like steam explosion, the pressureis then rapidly reduced to atmospheric levels, boiling the ammonia andexploding the lignocellulosic material. AFEX pretreatment appears to beespecially effective for biomass with a relatively low lignin content,but not for biomass with high lignin content such as newspaper or aspenchips (Sun and Cheng, 2002, Bioresource Technol., 83:1-11).

Concentrated or dilute acids may also be used for pretreatment oflignocellulosic biomass. H₂SO₄ and HCl have been used at highconcentrations, for instance, greater than 70%. In addition topretreatment, concentrated acid may also be used for hydrolysis ofcellulose (Hester et al., U.S. Pat. No. 5,972,118). Dilute acids can beused at either high (>160° C.) or low (<160° C.) temperatures, althoughhigh temperature is preferred for cellulose hydrolysis (Sun and Cheng,2002, Bioresource Technol., 83:1-11). H₂SO₄ and HCl at concentrations of0.3 to 2% (wt/wt) and treatment times ranging from minutes to 2 hours orlonger can be used for dilute acid pretreatment.

Other pretreatments include alkaline hydrolysis (Qian et al., 2006,Appl. Biochem. Biotechnol., 134:273; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol., 59:618), oxidative delignification, organosolvprocess (Pan et al., 2005, Biotechnol. Bioeng., 90:473; Pan et al.,2006, Biotechnol. Bioeng., 94:851; Pan et al., 2006, J. Agric. FoodChem., 54:5806; Pan et al., 2007, Appl. Biochem. Biotechnol.,137-140:367), or biological pretreatment.

Some of the pretreatment processes described above include hydrolysis ofthe hemicellulose and cellulose to monomer sugars. Others, such asorganosolv, prepare the substrates so that they will be susceptible tohydrolysis. This hydrolysis step can in fact be part of the fermentationprocess if some methods, such as simultaneous saccharification andfermentation (SSF), is used. Otherwise, the pretreatment may be followedby enzymatic hydrolysis with cellulases.

A cellulase may be any enzyme involved in the degradation oflignocellulose to glucose, xylose, mannose, galactose, and arabinose.The cellulolytic enzyme may be a multicomponent enzyme preparation,e.g., cellulase, a monocomponent enzyme preparation, e.g.,endoglucanase, cellobiohydrolase, glucohydrolase, beta-glucosidase, or acombination of multicomponent and monocomponent enzymes. Thecellulolytic enzymes may have activity, e.g., hydrolyze cellulose,either in the acid, neutral, or alkaline pH-range.

A cellulase may be of fungal or bacterial origin, which may beobtainable or isolated from microorganisms which are known to be capableof producing cellulolytic enzymes. Examples of such microbes aredescribed herein. Useful cellulases may be produced by fermentation ofthe above-noted microbial strains on a nutrient medium containingsuitable carbon and nitrogen sources and inorganic salts, usingprocedures known in the art.

Examples of cellulases suitable for use in the present inventioninclude, for example, CELLUCLAST (available from Novozymes A/S) andNOVOZYME (available from Novozymes A/S). Other commercially availablepreparations including cellulase which may be used include CELLUZYME,CEREFLO and ULTRAFLO (Novozymes A/S), LAMINEX and SPEZYME CP (GenencorInt.), and ROHAMENT 7069 W (Rohm GmbH).

The hydrolysis/fermentation of lignocellulosic materials may, andtypically does, require addition of cellulases (e.g., cellulasesavailable from Novozymes A/S). Typically, cellulase enzymes may be addedin amounts effective from 5 to 35 filter paper units of activity pergram of substrate, or 0.001% to 5.0% wt. of solids. The amount ofcellulases appropriate for the hydrolysis may be decreased by using agenetically modified microbe described herein. For instance, agenetically modified microbe that expresses a pectinase and a cellulase,such as cellobiase, will degrade polysaccharides such as pectin andcellobiose (a glucose disaccharide formed during saccharification) toresult in substrate for the genetically modified microbe to producedesirable metabolic products, thus requiring addition of less cellulasescompared to the same microbe without the modifications. The amount ofcellulases (e.g., cellulases available from Novozymes A/S) required forhydrolysis of the pretreated lignocellulosic material may be decreasedby at least 5%, at least 10%, at least 15%, at least 20%, at least 25%,or at least 30%. This decreased need for cellulases can result in asignificant decrease in costs associated with producing metabolicproducts from lignocellulosic materials.

The steps following pretreatment, e.g., hydrolysis and fermentation, canbe performed separately or simultaneously. Conventional methods used toprocess the lignocellulosic material in accordance with the methodsdisclosed herein are well understood to those skilled in the art.Detailed discussion of methods and protocols for the production ofethanol from biomass are reviewed in Wyman (1999, Annu. Rev. EnergyEnviron., 24:189-226), Gong et al. (1999, Adv. Biochem. Engng. Biotech.,65: 207-241), Sun and Cheng (2002, Bioresource Technol., 83:1-11), andOlsson and Hahn-Hagerdal (1996, Enzyme and Microb. Technol.,18:312-331). The methods of the present invention may be implementedusing any conventional biomass processing apparatus (also referred toherein as a bioreactor) configured to operate in accordance with theinvention. Such an apparatus may include a batch-stirred reactor, acontinuous flow stirred reactor with ultrafiltration, a continuousplug-flow column reactor (Gusakov, A. V., and Sinitsyn, A. P., 1985,Enz. Microb. Technol., 7: 346-352), an attrition reactor (Ryu, S. K.,and Lee, J. M., 1983, Biotechnol. Bioeng., 25: 53-65), or a reactor withintensive stirring induced by an electromagnetic field (Gusakov, A. V.,Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Appl. Biochem. Biotechnol., 56: 141-153). Smaller scale fermentationsmay be conducted using, for instance, a flask or a fleaker.

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHE), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC). The fermentation can be carried out by batch fermentation or byfed-batch fermentation.

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the coferementation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog., 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbiol. Mol. Biol. Reviews, 66: 506-577).

The final step may be recovery of the metabolic product. The methoddepends upon the metabolic product that is to be recovered, and methodsfor recovering metabolic products resulting from microbial fermentationof lignocellulosic material are known to the skilled person and usedroutinely. For instance, when the metabolic product is ethanol, theethanol may be distilled using conventional methods. For example, afterfermentation the metabolic product, e.g., ethanol, may be separated fromthe fermented slurry. The slurry may be distilled to extract theethanol, or the ethanol may be extracted from the fermented slurry bymicro or membrane filtration techniques. Alternatively the fermentationproduct may be recovered by stripping.

Also provided herein are methods for using the polypeptides describedherein. The methods typically include contacting a lignocellulosicmaterial with a polypeptide described herein, such as a PelApolypeptide, a PelB polypeptide, or a fragment thereof, under conditionssuitable for the degradation of pectin. The conditions may be alkaline,such as pH 9 to 10.5. The polypeptide may be used in combination withother carbohydrate degrading enzymes, such as arabinanase and/orxyloglucanase, as well as other pectinases. The polypeptides describedherein are useful in processing of materials, such as the pretreatmentof lignocellulosic material to prepare for the production of metabolicproducts, decreasing viscosity of solutions containing pectin,clarifying solutions such as fruit juices, retting and/or degumming offiber crops such as hemp, flax, or linen, treatment of pecticwastewater, production of Japanese paper, paper making, and oilextraction from oil-rich plant material, such as soy-bean oil fromsoy-beans, olive-oil from olives or rapeseed-oil from rape-seed orsunflower oil from sunflower (Hoondal et al., 2002, Appl. Microbiol.Biotechnol., 59:409-418, Kashyap et al., 2001, Bioresour. Technol.,77:215-227). The polypeptides described herein may be used for thepreparation of fibers or for cleaning of fibers, typically incombination with detergents. Cotton fibers consist of a primary cellwall layer containing pectin and a secondary layer containing mainlycellulose (Andersen et al., U.S. Pat. No. 7,273,745). During cottonpreparation or cotton refining part of the primary cell wall may beremoved. The polypeptides disclosed herein may be used as an aid duringcotton refining by removal of the primary cell wall or during cleaningof the cotton to remove residual pectic substances and prevent grayingof the textile.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

Example 1

Ethanologen Escherichia coli KO11 was sequentially engineered to producethe Klebsiella oxytoca EnzymeII^(cellulose) and phospho-βglucosidasegenes (casAB) as well as a pectate lyase (pelE) from Erwiniachrysanthemi, yielding strains LY40A (casAB) and JP07 (casAB; pelE),respectively. To obtain effective secretion of PelE, the Sec-independentpathway out genes from E. chrysanthemi on the cosmid pCPP2006 wereprovided to strain JP07 to construct strain JP07C. E. coli strainsLY40A, JP07, and JP07C possessed significant cellobiase activity in celllysates, while only strain JP07C demonstrated extracellular pectatelyase activity. Fermentation with sugar beet pulp at very low fungalenzyme loads during saccharification revealed significantly higherethanol production for LY40A and JP07C compared to KO11. While JP07Cethanol yields were not considerably higher than LY40A, investigation ofoligogalacturonide polymerization showed an increased breakdown ofbiomass to small chain (degree of polymerization ≦6)oligogalacturonides. Further engineering of E. coli JP07C to expressOgl, an oligogalacturonide lyase also from E. chrysanthemi, achievedeven further breakdown of polygalacturonate to monomeric sugars and leadto higher ethanol yields.

Materials and Methods

Bacterial strains and media. Bacterial strains, plasmids, andoligonucleotides used in this study are listed in Table 1. E. colistrains were grown at 37° C. in Luria-Bertani (LB) medium supplementedwith 2% wt/vol glucose for ethanologenic strains. When indicated,antibiotics were used at the following concentrations unless otherwisestated: chloramphenicol (Cm), 40 mg/L; ampicillin (Ap), 50 mg/L;kanamycin (Ku), 40 mg/L; erythromycin (Em), 150 mg/L; and spectinomycin(Spc), 50 mg/L. For enzyme assays, ethanologenic E. coli were grown inminimal media (MM) (Atlas, et al., 1993, Handbook of MicrobiologicalMedia. CRC Press, Inc., Boca Raton, Fla.) [0.02 M (NH₄)₂SO₄, 0.01 Msodium citrate, 8 mM Na₂PO₄, 2 mM MgSO₄.7H₂O, 1 mM KCl, 30 nMFeSO₄.7H₂O] with 0.5% wt/vol glucose and either 0.5% wt/volpolygalacturonic acid or cellobiose. All chemicals were obtained fromSigma Chemical Co. (St. Louis, Mo.). Oligonucleotides were synthesizedby Integrated DNA Technologies (Coralville, Iowa). Restriction enzymesand T4 DNA ligase were obtained from New England BioLabs (Ipswich,Mass.). DNA sequencing reactions were performed at the Sequencing andSynthesis Facility at the University of Georgia (Athens, Ga.).

TABLE 1 E. coli strains and plasmids used in this study.Strain, plasmid, or Source or  oligonucleotide Relevant characteristicsreference Escherichia coli KO11 pdc⁺ adhB⁺; Cm^(r) A LY40AKO11 with casAB This study JP07 LY40A with pelE; Cm^(r) Ap^(r)This study JP07C JP07 with pCPP2006; Cm^(r) Ap^(r) Spc^(r) This studyPlasmids pLOI1998 casR^(l) AB B pST76-K Ts; Cm^(r) C pLOI2708pST76-K derivative, lacY: casAB: lacA,Zm promoter This study pPEL748pelE D pLOI2090 pelE; Ap^(r) This study pCPP2006 out genes; Spc^(r) EpDMA160 pEVS107 derivative, mini-Tn7; mob; Em^(r) Kn^(r) E. V. StabbpEDH24 pDMA160 derivative, consensus promoter This study pEDH25pEDH24 derivative, pelE; Ap^(r) This study pUXBF13R6K ori; tns genes; Ap^(r) F pEVS104pRK2013 derivative; conjugal tra and trb genes G Oligonucleotides LPY15′-GAGATCTTAAGGAAAAACAGCATGGA-3′ (SEQ ID N0: 7) This study LPY25′-ATAGCCGGCGTCCAGAAT-3′ (SEQ ID NO: 8) This study LacYF5′-TTGCTCTTCCATGTACTATTTAAAAAACACAAAC-3′(SEQ ID NO: 9) Sigma GenosysLacYR 5′-TTGCTCTTCGTTAAGCGACTTCATTCACCTGAC-3′ (SEQ ID NO: 10)Sigma Genosys LacAF 5′-TTGCTCTTCCATGCCAATGACCGAAGAATAAGAG-3′(SEQ ID NO: 11) Sigma Genosys LacAR5′-TTGCTCTTCGTTAAACTGACGATTCAACTTTATA-3′ (SEQ ID NO: 12) Sigma GenosysLacZ 5′-GGTGAAGTGCCTCTGGATGT-3′ (SEQ ID NO: 13) This study CasA5′-CGCCTACCCGAGTGAGAATA-3′ (SEQ ID NO: 14) This study CasB5′-GCAAAGCGGAAGTCTACCAG-3′ (SEQ ID NO: 15) This study CynX5′-ATGCCTTCGGTGATTAAACG-3′ (SEQ ID NO: 16) This study Promoter5′-CTAGTTGACATGATAGAAGCACTCTACTATATT-3' (SEQ ID NO: 17) E. V. Stabb3′-AACTGTACTATCTTCGTGAGATGATATAACTAG-5′ (SEQ ID NO: 18) EDH1605′-TGCTCAACGGGAATCCTGCTCT-3′ (SEQ ID NO: 19) This study EDH2090F5′-GCGCATGGGCCCCACACAGGAAACAGCTATGACC-3′ (SEQ ID NO: 20) This studyEDH2090R 5′-GCATGCGGGCCCGTTACCAATGCTTAATCAGTGAGG-3′ (SEQ ID NO: 21)This study EDHPelB 5′-TCAGCACGAACACGAACCGTCTTA-3′ (SEQ ID NO: 22)This study EDHPelE 5′-TGTGCTGCAAGGCGATTAAGTTGG-3′ (SEQ ID NO: 23)This study A, Ohta et al., 1991, Appl. Environ. Microbiol., 57: 893-900;B, Lai et al., 1997, Appl. Environ. Microbiol. 63: 355-363; C, Posfai etal., 1997, J. Bacteriol., 179: 4426-4428; D, Keen et al., 1986, J.Bacteriol., 168: 595-606; E, He et al., 1991, Proc. Natl. Acad. Sci.,88: 1079-1083; F, Bao et al., 1991, Gene, 109: 167-168; G, Stabb et al,.2002, Methods Enzymol., 358: 413-426.

Genetic procedures and recombinant techniques. Standard methods wereemployed to construct plasmids and transfer DNA (Sambrook, et al., 1989.Molecular cloning: a laboratory manual, 2 ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). PCR was performed using eitherPlatinum Taq (Invitrogen, Carlsbad, Calif.) or Phusion™ High-FidelityDNA Polymerase Kit (New England BioLabs, Ipswich, Mass.), following themanufacturer's recommendations for reaction programs.

Chromosomal insertion of K. oxytoca casAB genes in E. coli KO11. ThecasAB genes from K. oxytoca (Lai, et al., 1997, Appl. Environ.Microbiol. 63:355-363) were chromosomally integrated into E. coli KO11(Ohta, et al., 1991. Appl Environ Microbiol 57:893-900), between thelacY and lacA genes after adding a strong surrogate promoter, (Zhou, etal., 1999, J. Ind. Microbiol. Biotechnol. 22:600-607 (FIG. 1). The DNAfragment constructed for integration has been deposited in GenBank(Accesssion No. EU848570). Primers used in construction are listed inTable 1 (LPY1, LPY2, LacYF, LacYR, LacAF, and LacAR)

For chromosomal insertion, the casAB genes were amplified from pLOI1998(Lai, et al., 1997, Appl. Environ. Microbiol. 63:355-363) and ultimatelyengineered into pLOI2707, a temperature conditional vector, with lacYand lacA flanking the casAB genes. Z. mobilis genomic DNA was randomlyinserted upstream of casAB on pLOI2707 to create a library; these cloneswere screened for large colony size and dark red color on MacConkey agarplates with 2% wt/vol cellobiose to find a strong surrogate promoter forcellobiose utilization. One plasmid, designated pLOI2708, whichcontained an insert of approximately 1 kb with a promoter, was chosenfor further study. After electroporating E. coli K011 with pLOI2708 andselecting for casAB recombinants, cells were screened for red colonycolor on MacConkey agar containing 2% wt/vol cellobiose and LB agarcontaining 2% wt/vol glucose and 600 mg/L chloramphenicol to select forhigh expression of casAB and Z. mobilis pdc and adhB, respectively. Thestrain generated was named E. coli LY40A.

Chromosomal insertion of E. chrysanthemi pelE gene in E. coli LY40A. Adouble-stranded E. coli consensus promoter sequence was constructed byheating two complementary single-stranded DNA oligos with overhangs at98° C. for 10 min (Table 1). The oligos were cooled to room temperature,cloned into the AvrII site of pDMA160 to make pEDH24, and transformedinto E. coli BW23474 using a standard heat shock protocol (Sambrook, etal., 1989. Molecular cloning: a laboratory manual, 2 ed. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). Directionality of thepromoter was confirmed via DNA sequencing using the EDH160 primer. TheBsrBI fragment carrying the pelE gene from pPEL748 (Keen, et al. 1986.,J. Bacteriol. 168:595-606) was inserted into the SmaI-PstI site of pUC18to generate pLOI2090. The pelE and bla genes were amplified frompLOI2090 via PCR using primers EDH2090F and EDH2090R with engineeredApaI sites and cloned into pEDH24 at the ApaI site. Subsequent cloneswere investigated for directionality of the pelE-bla fragment, and theplasmid with pelE-bla in the correct orientation to the consensuspromoter was named pEDH25. A triparental mating of E. coli LY40A with E.coli BW23474 pUXBF13), E. coli BW23473 pEVS104 (Stabb, et al., 2002,Methods Enzymol. 358:413-426), and E. coli BW23474 pEDH25, was performedto insert the mini-Tn7 transposon with pelE and bla into the chromosome,yielding strain E. coli JP07 after selection on LB containing Cm and Ap.Strain verification was accomplished by sequence analyses using primersEDHPelB and EDHPelE. Cosmid pCPP2006 (He, et al., 1991, Proc. Natl.Acad. Sci. 88:1079-1083) was transformed into E. coli JP07 usingstandard heat shock protocol (Sambrook, et al., 1989. Molecular cloning:a laboratory manual, 2 ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.), giving strain E. coli JP07C.

Construction of E. coli JP08C. To construct pTOGL, theoligogalacturonide lyase gene, ogl, was PCR amplified from Erwiniachrysanthemi 3937 using primers OglF and OglR and cloned into pCR2.1using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, Calif.). OglFcontained the consensus E. coli promoter sequence employed in previousexperiments. pTOGL was then transformed into JP07C via heat shock(Sambrook, et al., 1989. Molecular Cloning: a Laboratory Manual, 2ndedition), giving strain E. coli JP08C.

Cellobiase assay. Assays for cellobiase activity were performedessentially as described previously (Moniruzzaman, et al., 1997, Appl.Environ. Microbiol. 63:4633-4637). Briefly, ethanologenic E. coli weregrown in LB with 2% wt/vol cellobiose for 24 hours at 37° C. withshaking. Cells were harvested via centrifugation at 10,000 g for 10minutes and lysed by sonication in 50 mM phosphate buffer, pH 7.2.Lysates were assayed for 15 minutes in 50 mM phosphate buffer with 2 mMp-nitrophenyl-β-D-1,4-glucopyranoside (PNPG). The reaction wasterminated by the addition of 1 M Na₂CO₃ and ρ-nitrophenol content wasmeasured at 410 nm. Units are defined as μmol product formed per minuteper mL. Protein assays were performed on the supernatant by the Bradfordmethod (Bradford, 1976, Anal. Biochem. 72:248-254), and enzyme activityreported as specific activity in U/mg protein. Data represents the meanof three separate experiments.

Pectate lyase assay. Assays for pectate lyase activity were performed asdescribed previously (Collmer, et al., 1988, Methods in Enzymology, vol.161. Academic Press, Inc., San Diego, Calif.). Briefly, ethanologenic E.coli were grown in MM with 0.5% wt/vol glucose and 0.5% wt/volpolygalacturonic acid for 48 hours at 37° C. with shaking. Culturesupernatant was harvested via centrifugation at 10,000 g for 10 minutes.Supernatant was assayed by rapidly mixing with substrate [60 mMTris-HCl, pH 7.2, 0.6 mM CaCl₂, 0.24% wt/vol polygalacturonic acid],both previously equilibrated to 37° C., and monitoring the formation of4,5-unsaturated products at 232 nm for 5 min with a linear rate ofreaction for at least 30 s. Units are defined as μmol product formed permin per mL. Protein assays were performed on the supernatant by theBradford method (Bradford, M. 1976, Anal. Biochem. 72:248-254), andenzyme activity reported as specific activity in U/mg protein. Datarepresents at least four separate experiments.

Sugar beet fermentations and analysis of ethanol production and reducedsugars. Fermentations were performed essentially as described previously(Doran, et al., 2000, Appl. Biochem. Biotechnol. 84-86:141-152). Sugarbeet pulp dry weight was calculated using a Denver Instrument IR 35Moisture Analyzer (Denver, Colo.). In a blender, 10 g dry wt sugar beetpulp, 100 mL of 2×LB liquid media, and water to a final volume of 200 mLwere blended at full speed for 10 s and then autoclaved in a 500 mLfleaker; blending was necessary to reduce particle size as very lowfungal enzymes loads were used. The fleakers were placed in a water bathat 45° C. and mixed with magnetic stirrers. The pH was adjusted to 4.5using a Jenco 3671 pH controller (San Diego, Calif.). Spezyme CP(Genencor; Copenhagen, Denmark) and pectinase from Aspergillus niger(Sigma P2736) (Novozymes; Franklinton, N.C.) were added to the fleakerat concentrations of 0.5 filter paper units (FPU) per g dry wt and 4polygalacturonase units (PGU) per g dry wt, respectively. After 24 h,the pH was increased to 6.8 and the temperature was decreased to 35° C.and maintained throughout the fermentation. Appropriate antibiotics wereadded to each fleaker, and they were inoculated to an OD₅₅₀ 1.0 withcells collected via centrifugation (10 000×g; 10 min) from overnightcultures of E. coli strains KO11, LY40A, JP07C, or JP08C. Fermentationswere run for 72 h with samples collected every 24 h.

To quantify ethanol production, gas chromatography (GC) was performed;fermentation supernatant samples were filtered with a 0.22 μm filterprior to analysis. Ethanol concentrations were normalized to zero toaccount for ethanol added from antibiotic stocks. Reducing sugaranalysis was performed using the dinitrosalicylic acid assay method(Miller, et al., 1959, Anal. Chem. 31:426-428).

Examination of oligogalacturonides. To quantify oligogalacturonides witha degree of polymerization (dp) less than 6, fermentation supernatantwas diluted 1:3 in water and ethanol was added to a final concentrationof 11% (vol/vol). The solution was incubated with agitation for 16 h at4° C. and then centrifuged at 7500 g for 15 min. This supernatant wasdiluted and analyzed at 235 nm (Spiro, et al., 1993. Carbohydr. Res247:9-20). The absorbance of fermentation supernatant preparation of E.coli KO11 at 72 hours was used as the baseline. Data represents theaverage of two experiments.

Results and Discussion

Construction of E. coli LY40A. Previous research identified cellobiosephosphoenolpyruvate-dependent phosphotransferase genes (casAB) from K.oxytoca that allowed rapid growth of E. coli DH5α with cellobiose as thesole carbon source (Lai, et al., 1997. Appl. Environ. Microbiol.63:355-363). However, when a plasmid containing casAB was transferred toE. coli K011, expression was poor; mutational studies of this plasmid inK011 suggested the native promoter was more tightly controlled in thisstrain (Moniruzzaman, et al., 1997, Appl. Environ. Microbiol.63:4633-4637). To create a stable, cellobiose-fermenting strain of E.coli K011, the casAB genes were inserted into the chromosome with astrong surrogate promoter. The strain generated was named E. coli LY40A.Enzyme assays with p-nitrophenyl-β-D-1,4-glucopyranoside verified theabsence and presence of cellobiase activity in K011 and LY40A,respectively (Table 2). Integration by double homologous recombinationwas verified using primers (LacZ, CasA, CasB, and CynX) that includedthe lacZ and cynX genes flanking the genomic insertion site (Table 1).

TABLE 2 Cellobiase and extracellular pectate lyase specific activity forE. coli KO11 and derivative strains (standard deviation; n = 3) E. coliSpecific Activity (IU/mg protein) Strain Cellobiase Pectate Lyase KO11 00 LY40A 15.0 ± 0.4 0 JP07 15.8 ± 1.0  0.2 ± 0.3 JP07C 15.3 ± 1.1 18.9 ±1.2 JP08C  5.4 ± 0.3 49.3 ± 0.9

While chromosomal insertion of casAB improves E. coli K011 by enablingbreakdown of cellobiose without supplemental cellobiase, the complexityof lignocellulosic substrates necessitates many other types of enzymesfor breakdown. Further engineering of E. coli LY40A with additionaltypes of enzymes should therefore enable decreased use of exogenousenzymes.

Construction of E. coli JP07 and JP07C. In lignocellulosic substrates,pectin interacts with lignin, hemicellulose, and cellulose, anddegradation of pectin is necessary to allow the disintegration of othercomponents. Therefore, a pectate lyase, which cleaves thepolygalacturonate repeating chains of pectin, was engineered into E.coli LY40A with a surrogate promoter.

For chromosomal integration, a mini Tn7 system was used, which insertsas a single copy in the neutral att site in the E. coli chromosome (Bao,et al., 1991. Gene 109:167-168). pelE and bla were PCR amplified frompLOI2090 and cloned into pDMA160 with an E. coli consensus promoter,resulting in plasmid pEDH25. The plasmid was sequenced to verifypromoter directionality and pelE sequence; a 61 bp deletion occurredbetween the promoter and pelE, but did not affect expression (data notshown). pEDH25 was conjugated into E. coli LY40A, and pelE transposedinto the att site resulting in strain E. coli JP07.

Previous studies with E. chrysanthemi pectate lyases showed that aSec-independent pathway, encoded by the out genes, was necessary forsecretion of these enzymes (He, et al., 1991, Proc. Natl. Acad. Sci.88:1079-1083). A cosmid with a 40 kb fragment of the E. chrysanthemigenome containing the out genes, pCPP2006, was electroporated intostrain E. coli JP07 to give strain K coil JP07C (He, et al., 1991, Proc.Natl. Acad. Sci. 88:1079-1083). Enzyme assays withp-nitrophenyl-β-D-1,4-glucopyranoside were performed to ensure thatcellobiase activity in E. coli JP07 and JP07C was not affected by theaddition of pelE (Table 2). Subsequently, assays were performed withpolygalacturonic acid to demonstrate extracellular pectate lyaseactivity (Table 2). E. coli KO11 and LY40A demonstrated no activity,while JP07 varied greatly, reaching, at the most, 0.5 U/mg protein; theoccasional presence of activity could be attributed to cell lysis. E.coli JP07C, however, exhibited 18.9 U/mg protein of extracellularpectate lyase activity, demonstrating the functionality of the out genessecretion system.

Comparison of E. coli K011, LY40A, and JP07C. To demonstrate use ofthese engineered E. coli strains, sugar beet pulp fermentations wereperformed with very low fungal enzyme loads during pretreatment (FIG.2A). A typical fermentation of sugar beet pulp with E. coli KO11 wouldbe performed with 10.5 FPU/g dry wt cellulase, 120.4 PGU/g dry wtpectinase, and 6.4 CBU/g dry wt cellobiase (Doran, et al., 2000, hit.Sugar. J. 102:336-340). To determine the effect of the engineeredenzymes, 0.5 FPU/g dry wt cellulase and 4 PGU/g dry wt polygalacturonasewere used; with such low loads of exogenous enzymes, only a smallportion of the lignocellulose structure is degraded, which sequestersmuch of the sugar available for conversion to ethanol and leads to lowethanol yields. Both E. coli LY40A and JP07C had significantly higherethanol yields than E. coli KO11. Examination of reducing sugarsdemonstrates E. coli KO11's low yield: the high amount (140-185 μg/mL)of reducing sugars present throughout the fermentation corresponds tooligomeric substrates the strain is unable to metabolize. Comparisonwith casAB-containing E. coli LY40A and JP07C, whose reducing sugarconcentrations decrease to near zero within 24 hours, suggests a majorcomponent of the reducing sugars which E. coli KO11 is unable to consumeis cellobiose, illustrating the significance of the addition of casAB tothe strain.

Ethanol yields for E. coli JP07C were not significantly higher thanthose for LY40A. However, the concentration of reducing sugars for E.coli JP07C continually increased after 24 h while that of LY40A did not(FIG. 2A). If PelE produced by E. coli JP07C is cleaving largepolygalacturonate chains without releasing large amounts of monomericsugars, the reducing sugar concentration would increase while ethanolproduction would not. To test this hypothesis, oligogalacturonides witha degree of polymerization (dp) greater than six were precipitated fromthe fermentation samples and the remaining oligogalacturonides with a dpof six or less were measured by absorbance at 235 nm. As seen in FIG.2B, the absorbance of E. coli JP07C is significantly higher than that ofKO11 or LY40A throughout the fermentation, and, after fermentation ofsugars released from the fungal enzymes, continues to increase from 24to 72 h; this difference in absorbance corresponds to an increase ofshort chain oligogalacturonides throughout fermentation, demonstratingthe enzymatic breakdown of polygalacturonate.

Comparison of JP07C and JP08C. The oligogalacturonide lyase of Erwiniachrysanthemi 3937 (Collmer, et al., 1981, Proc. Natl. Acad. Sci.78:3920-3924) was transformed into E. coli JP07C to give strain JP08C,where ogl is maintained on plasmid pTOGL. Enzyme assays with JP08Cdemonstrated a large increase in the production of 4,5-unsaturatedproducts, indicating ogligogalacturonide activity in addition to pectatelyase activity (Table 2). Sugar beet pulp fermentations were performedto determine if this Ogl activity leads to higher ethanol yields thanthat of predecessor strains. The combination of pelE and oglsignificantly increased ethanol production when compared to LY40A (FIG.3). Examination of reducing sugar concentrations for JP08C shows thatwhile they are decreased in comparison to JP07C, they do continue toincrease slightly throughout the fermentation; this suggests thatpolygalacturonic acid chains are being released from the sugar beetpulp, but are not being cleaved into di- and tri-galacturonides subjectto oligogalacturonide lyase activity. As sugar beet pulp is highlymethyl esterified (60%), the activities of both PelE and Ogl might bepartially inhibited, and further addition of a pectin methylesterasecould increase the activity of these two enzymes (Sun, et al., 1998,Polymer J. 30:671-677).

Engineering these ethanologenic E. coli strains to producelignocellulose degrading enzymes during fermentation can allow partialsaccharification and co-fermentation, which enables decreased use ofexogenous fungal enzymes in biomass saccharification steps, reducing thecost of the entire process. The addition of casAB for cellobioseutilization significantly impacts ethanol production fromlignocellulosic biomass and drastically reduces the need for fungalcellobiases, possibly eliminating the need for this type of enzymealtogether. While the addition of pelE did not display the same effect,secretion of pectate lyase did considerably increase degradation ofpolygalacturonate, and further engineering of E. coli JP07C to producean oligogalaturonate lyase, ogl, allowed breakdown of polygalacturonateto monomeric sugars during fermentation and increased ethanol yield.Engineering of E. coli JP08C demonstrates the possibility of creating astrain of E. coli for consolidated bioprocessing, thereby eliminatingthe need for exogenous enzymes altogether (Lynd, et al., 2008, NatureBiotechnol. 26:169-172). Further work to integrate cellulases,hemicellulases, and other pectinases will advance this goal of a singlemicroorganism capable of both degradation and fermentation oflignocellulosic biomass.

Example 2

Paenibacillus amylolyticus C27 was isolated from the hindgut of Tipulaabdominalis and found to produce lignocellulose-degrading enzymes. Alibrary was constructed with C27 genomic DNA for heterologous expressionof biological characteristics in Escherichia coli. Two pectate lyasegenes, pelA and pelB, were identified while screening a genomic libraryin E. coli for pectinase activity. PelA encodes a 222 amino acid proteinand demonstrated highest activity on polygalacturonic acid, but retained60% and 56% of maximum activity on 8.5% and 90% methylated pectin,respectively. CaCl₂ was required for activity, and optima were pH 10.5,45° C., and 1.5 mM CaCl₂. PelA has high identity (95%) to PelA from P.barcinonensis, and is a subclass of the pectate lyase family III fromsaprophytic, non-pathogenic bacteria. On the other hand, pelB encodes a392 amino acid protein. Although PelB showed the highest activity on20-34% methylated pectin, it retained 67%, 51%, 25%, and 1% of itsmaximum activity on polygalacturonic acid, 8.5%, 55-70%, and 90%methylated pectin, respectively. The optima were pH 9.5, 55° C., and 0.5mM CaCl₂, and CaCl₂ was required for the enzymatic activity. PelB showsno significant similarity to any known enzyme, but contains manyconserved sites of the pectate lyase family I subclass. It shows highestamino acid identity of only 28% to Bacillus sp. YA-14 PelK, B.licheniformis ATCC 14580 Pel, and B. subtilis reference strain 168 Pel.

Materials and Methods

Bacterial strains and plasmids. P. amylolyticus C27 was isolated fromthe hindgut of Tipula abdominalis (Cook, et al., 2007, Appl. Environ.Microbiol., 73, 5683-5686) and grown as described previously (Henriksenet al., 2007, Lett. Appl. Microbiol., 45, 491-496) in either tryptic soybroth or Davis minimal media. Strains and plasmids used for cloning arelisted in Table 1. Escherichia coli strains were grown at 37° C. inLuria Bertani (LB) broth with 50 mg L⁻¹ ampicillin (Ap), whereindicated.

TABLE 1 Cloning strains and plasmids used in this studyStrain, plasmid, or Source or oligonucleotide Relevant characteristicsreference Escherichia coli DH5αF⁻endA1glnV44thi-1recA1relA1gyrA96 deoR nupG AΦ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r_(K) ⁻ m_(K) ⁺), λ- PlasmidspUC19 lacZα⁺; Ap^(r) Invitrogen (Carlsbad, CA) pEDH13C2pUC19 derivative; P. amylolyticus DNA fragment with pelA This studypEDH27 pUC19 derivative; pelA⁺ This study pUC19-19F6pUC19 derivative; P. amylolyticus DNA fragment with pelB This studypWEB1 pUC19 derivative; pe1B⁺ This study Oligonucleotides Seq25′-ACACTGAACGAAATGCTCCAAACC-3′ (SEQ ID N0: 24) This study for Pel APLAscF 5′-GTACAGGGCCCGGATCCTTGACATGATAGAAGCACTCTACT This studyATATTCTAGTGCTTCTACGGTTCTGTGGGACAA-3′ (SEQ ID NO: 25) PLAscR5′-CGATCAAGCTTGGGCCCGAGCGGCCGCCTCGAGTCCACATG This studyGTTTGGAGCATTTCG-3′ (SEQ ID NO: 26) SCpelBF5′-GCAGTGAGCTCTTGACATGATAGAAGCACTCTACTATATTC This studyTAGTTATACTTATCGGGAGGAATCG-3′ (SEQ ID NO: 27) SCpelBR5′-CATGGGATCCCGGAGCGCTTAACTTAGTAACTC-3′ (SEQ ID NO: 28) This study Seq25′-ACCGCAGCATCGCTTATGTAGGTA-3′ (SEQ ID NO: 29) This study Seq35′-AGTATCACTGTTGCCACGGAAGGA-3′ (SEQ ID NO:30) This study Seq45′-TGGTGAGTCCATTAAAGCCGTCCA-3′ (SEQ ID NO:31) This study A, Meselson andYuan, 1968, Nature, 217: 1110-1114.

Library construction and enzymatic screening. Genomic DNA from P.amylolyticus C27 was prepared using the DNeasy Blood and Tissue Kit(Qiagen, Valencia, Calif.). After partial digestion with Sau3AI andagarose gel extraction of 2-5 kb fragments, P. amylolyticus C27 genomicfragments were ligated into BamHI digested pUC19, transformed into E.coli DH5α by heat shock (Sambrook, et al., 1989, Molecular cloning: alaboratory manual, Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory), and grown on LB agar with 50 mg/L ampicillin, 1 mg/L X-Gal,and 2.5 mg/L IPTG. Insert-containing transformants were screened forpectinase activity on polygalacturonase medium (Starr, et al., 1977, J.Clin. Microbiol., 6, 379-386). After growth, plates were flooded with 2NHCl and pectinase-producing colonies were identified by the appearanceof clearing surrounding colonies.

Pectinase identification and subcloning. At the Sequencing and SynthesisFacility at the University of Georgia, plasmid pEDH13C2 from apectinase-producing clone (13C2) was first sequenced with primer M13Ffollowed by primer Seq2 Primers. PLAscF and PLAscR were used to subclonethe pectinase gene, pelA, from pEDH13C2 into pUC19 using BamHI andHindIII sites, which were engineered into the forward and reverseprimer, respectively, giving plasmid pEDH27. The forward primer alsocontained a consensus E. coli promoter (E. V. Stabb). Plasmid pUC19-19F6from another pectinase-producing clone (19F6) was sequenced with primer19F6 M13R followed by Seq 2, Seq3, Seq4. SCpelBF and SCpelBR were usedto subclone the pectinase gene, pelB, from pUC19-19F6 into pUC19 usingSacI and BamHI restriction enzyme sites that were engineered into theforward and reverse primers, respectively, creating plasmid pWEB1. Theforward primer contained the same consensus E. coli promoter (E. V.Stabb) as the forward primer used to create pEDH27.

Enzyme assays. PelA pectate lyase assays were performed essentially asdescribed (Collmer, et al., 1988, Methods in Enzymology. San Diego,Calif., Academic Press, Inc.) (Soriano, et al., 2000, Microbiology, 146,89-95) with E. coli DH5α pEDH27 cell extracts prepared by sonication.The standard enzyme assay mixture for PelA contained 0.2% (w/v)polygalacturonic acid (PGA, Sigma) or pectin (8.5% esterified citruspectin MP Biomedicals (Irvine, Calif.) or 90% esterified citrus pectinpurchased from Sigma (St. Louis, Mo.)) in a final volume of 1 mL 50 mMglycine buffer pH 10.5 with 1.5 mM CaCl₂; the assay mixture and enzymepreparation were equilibrated to 45° C. and monitored for the formationof Δ-4,5-unsaturated products at 235 nm for 1 to 3 min.

Pectate lyase assays for PelB were performed similar to PelA but with E.coli DH5α pWEB1 supernatant. The standard enzyme assay mixture for PelBcontained 0.2% (w/v) polygalacturonic acid (PGA, Sigma) or pectin (8.5%esterified citrus pectin MP Biomedicals (Irvine, Calif.), 20-34%,55-70%, and 90% esterified citrus pectin Sigma (St. Louis, Mo.)) in afinal volume of 1 mL 50 mM glycine buffer pH 9.5 with 0.5 mM CaCl₂; theassay mixture and enzyme preparation were equilibrated to 55° C. andmonitored for the formation of Δ-4,5-unsaturated products at 235 nm for1 min. One unit of enzyme activity was defined as the amount of enzymethat produces 1 μmol 4,5-unsaturated product per minute under both assayconditions described. Specific activity is reported as U/mg protein andthe Bradford method was used to determine protein concentration ofenzyme preparations.

The pH optimum was determined at 40° C. with 1 mM CaCl₂ using thefollowing buffers over the stated pH ranges: 50 mM sodium citrate, pH3.0-4.0; 50 mM sodium acetate, pH 4.0-6.0; 50 mM sodium phosphate, pH6.0-8.0; 50 mM Tris-HCl, pH 8.0-9.0; and 50 mM glycine, pH 9.0-12.0. ForPelA the temperature optimum was determined at pH 10.5 in a range of25-55° C., and the CaCl₂ concentration optimum was determined at pH 10.5and 45° C. in a range of 0-2.5 mM. For PelB the temperature optimum wasdetermined at pH 9.5 in a range of 15-60° C., and the CaCl₂concentration optimum was determined at pH 9.5 and 55° C. in a range of0-2.5 mM.

Results and Discussion

Cloning and identification of the pectate lyase. A library containing 2-to 5-kb chromosomal fragments of P. amylolyticus C27 was constructed inE. coli DH5α. Two pectinase-positive clones, 13C2 and 19F6, wereidentified after screening approximately more than 6,000 clones.Sequencing of the plasmid carried in the 13C2 clone, pEDH13C2, showed aninsert of 2 kb. A single ORF of 669 bp was identified and named pelA. Aputative ribosomal-binding (AAGGGAGGA) site is located eight nucleotidesupstream of the ATG start codon; also upstream of pelA is a putativepromoter with −10 (TTGTAA) and −35 (TTCTGT) elements. The deducedprotein sequence of the ORF is 222 amino acids. The protein has anN-terminal region with features of a Bacillus signal peptide, and themost likely cleavage site is between amino acids 26 and 27 (Nielsen, etal., 1997, Protein Eng., 10:1-6).

Sequencing of the plasmid carried in the 19F6 clone, pUC10-19F6, showedan insert of 1.5-kb. A single ORF of 1176 bp was identified and namedpelB and encodes a 302 amino acid protein. Located eight nucleotidesupstream of the ATG start codon is a putative ribosomal-binding(GGGAGGAA) similar to a Shine-Dalgerno site. Also, located upstream ofpelB is a putative promoter with −10 (TATACT) and −35 (TTGTGA) elements.

PelA and PelB were compared to known proteins by performing aprotein-protein BLAST (blastp) using the NCBI database (Altschul, etal., 1997, Nucleic Acids Res., 25, 3389-3402). Homology was found forPelA to pectate lyases within family III (PL3), but not any other class.PelA was 95% identical to PelA from P. barcinonensis (Soriano, et al.,2000, Microbiology, 146, 89-95), but also showed high identity to otherBacillus sp. pectate lyases: 78% to Bacillus sp. KSM-P15 pectate lyase(Hatada, et al., 2000, Eur. J. Biohcem., 267, 2268-2275), 55% to B.subtilis PelC (Soriano et al., 2006, Microbiology, 152, 617-625), 54% toB. licheniformis YvpA, and 53% to Bacillus sp. P-2850 pectate lyase.PelA has lower identity to phytopathogens Fusarium solani PelB (31%)(Guo, et al., 1995, J. Bacteriol., 177, 7070-7077), Erwinia chrysanthemiPelI (15%) (Shevchik, et al., 1997, J. Bacteriol., 179, 7321-7330), andE. carotovora Pel3 (12%) (Liu, et al., 1994, Appl. Environ. Microbiol.,60, 2545-2552). All of these enzymes have an arginine residue (Arg-157in C27 PelA), which is believed to extract a proton during theβ-elimination mechanism of the reaction (Akita, et al., 2001, ActaCryst., D57, 1786-1792). Three of four signature blocks of conservedresidues for PL3 enzymes (Shevchik, et al., 1997, J. Bacteriol., 179,7321-7330) are found in PelA, but, like P. barcinonensis PelA, Bacillussp. KSM-P15 PL, B. subtilis PelC, B. licheniformisYvpA, and Bacillus sp.P-2850 PL enzymes, the fourth block of residues is not conserved; it isreplaced by another domain, not found in other pectate lyases (FIG. 4)(Soriano, et al., 2006, Microbiology, 152, 617-625). Additionally, theseenzymes have high homology to each other and lower cysteine content thanother family PL3 pectate lyases. PelA appears to belong to a subgroup offamily PL3 enzymes from saprophytic bacteria (Soriano, et al., 2006,Microbiology, 152, 617-625) which includes P. barcinonensis PelA,Bacillus sp. KSM-P15 PL, B. subtilis PelC, B. licheniformis YvpA, andBacillus sp. P-2850 PL.

PelB showed homology to family I pectate lyases (PL1), but not to anyother class. PelB showed highest identity of only 28% to Bacillus sp.YA-14 PelK (Kim, et al., 1994, Biotech. Biochem. 58, 947-949), B.licheniformis ATCC 14580 Pel (Rey, et al., 2004, Genome Biology, 5,R77), and B. subtilis reference strain 168 Pel (Kunst, F. et al. 1997,Nature, 390:249-256). In addition, PelB showed lower identity to otherpectate lyases: 27% to Thermotoga maritime PelA (Kluskens, et al., 2003,Biochem. J., 370, 651-659), and 26% to B. subtilis BS-2 Pel, and B.amyloliquefaciens TB-2 Pel. All of the enzymes listed above contain thecore structure of the parallel β-helix (vWIDH region), conservedcatalytlic sites, conserved calcium binding sites, and sites conservedin all thermostable PL1 pectate lyases (FIG. 5). On the other hand, PelBonly contains six of the seven conserved sites in all thermostable PL 1pectate lyases and does not contain any of the three conserved catalyticsites. PelB does contain the core structure of the parallel β-helix,vWIDH, but does not contain the other two pectate lyase conservedsequence patterns, AxDIKGxxxxVTxS and VxxRxPxxRxGxxHxxxxN (Xiao, et al.,2007, Appl. Environ. Microbiol.: 10:1-28). (Henrissat, et al., 1995,Plant Physiol., 107, 963-976). All of these enzymes, including PelB(Arg-157 in C27 Pel B), also have the arginine residue, like PelA, thatis thought to extract a proton during the β-elimination mechanism of thereaction (Akita et al., 2001, Acta Cryst., D57, 1786-1792). It appearsas though PelB is mostly likely a subclass of family I pectate lyase(PL1).

Characterization of P. amylolyticus C27 PelA and PelB. SDS-PAGE analysisof E. coli DH5α carrying plasmid pEDH27 cell extract showed a band ofapproximately 23 kDa (the predicted size of PelA) not present in theextract of E. coli DH5α pUC19 (data not shown). These extracts exhibitedpectate lyase activity on polygalacturonic acid (PGA), but did not showpolygalacturonase, xylanase, or cellulase activity usingdinitrosalycylic acid assays. PelA was active within a pH range of 7.5to 11.5, with optimal activity at pH 10.5 (FIG. 6A). The temperatureoptimum was 45° C., but PelA retained at least 50% of its activitywithin a range of 25 to 50° C. (FIG. 6B). CaCl₂ was necessary foractivity, as it is for all known pectate lyases (Jurnak et al., 1996, INVISSER, J. & VORAGEN, A. G. J. (Eds.) Pectin and Pectinases. Amsterdam,Elsevier), with maximum activity at 1.5 mM (FIG. 6C). The activity ofPelA on citrus pectin was also investigated. Assays with 20-34% and 90%methylesterified citrus pectin demonstrated activity at 60% and 56% ofthe maximum activity on PGA, respectively (FIG. 7).

The high activity of PelA on both PGA and pectins with low and highlevels of methylation is unusual, but was also observed for PelA from P.barcinonensis and PelC from B. subtilis (Soriano, et al., 2000,Microbiology, 146, 89-95) (Soriano, et al., 2006, Microbiology, 152,617-625. Other family PL3 enzymes, like PelB and PelC from E.chrysanthemi, are active on PGA, but have highest activity on pectinwith low levels of methylation with no activity on highly methylatedpectin (Tardy, et al., 1997, J. Bacteriol., 179, 2503-2511). Conversely,PelI from E. chrysanthemi and PelB from E. carotovora have highestactivity on 45% and 68% methylated pectin, respectively, and low or noactivity on PGA (Shevchik, et al., 1998, Mol. Microbiol., 29, 1459-1469)(Heikinheimo, et al., 1995, Mol Plant Microbe nteract., 8, 207-217).Thus, the P. amylolyticus C27 PelA, P. barcinonensis PelA, and B.subtilis PelC substrate utilization range, with activity on PGA as wellas pectin with any degree of methylation, are unique among the pectatelyases described to date (FIG. 7).

While highly similar, P. amylolyticus C27 PelA, P. barcinonensis PelA(Soriano, et al., 2000, Microbiology, 146, 89-95), and B. subtilis PelC(Soriano, et al., 2006, Microbiology, 152, 617-625) do have distinctdifferences in activity optima and substrate preference. While theoptima for C27 PelA is pH 10.5, both the P. barcinonensis PelA and PelCfrom B. subtilis have highest activity at pH 10 when assayed using thesame method. The temperature optima differ for all three enzymes: forC27 PelA, it is 45° C., P. barcinonensis PelA, 50° C., and B. subtilisPelC, 65° C. Likewise, activity on pectic substances differs; the B.subtilis PelC shows highest activity on 22% methylated pectin, P.barcinonensis PelA on PGA or 22% methylated pectin, and C27 PelA on PGA.

PelB from P. amylolyticus was active within a pH range of 7.5 to 10.5,but the optimal activity was at pH 9.5 (FIG. 8A). PelB showed greatestactivity from 40-55° C. The optimum temperature was 55° C., butsignificantly less activity was observed at 60° C. (FIG. 8B). PelB issimilar to all other pectate lyases, in that, it requires CaCl₂ in orderto be active (Jurnak, et al., 1996, IN VISSER, J. & VORAGEN, A. G. J.(Eds.) Pectin and Pectinases. Amsterdam, Elsevier). The optimum CaCl₂concentration was 0.5 mM, but PelB still retained more than 75% of itsactivity within a range of 1.0-2.0 mM, including 96% of its activity at1.5 mM (FIG. 8C).

Since PelB did not show high amino acid identity to any other knownenzymes, its ability to be active on PGA and methylated pectin wasstudied by running assays with a range of pectic substrates: PGA, 8.5%,20-34%, 55-70%, and 90% methylesterified citrus pectin. PelB showedhighest activity on 20-34% methylated pectin, but retained 67%, 51%,25%, and 1% of its maximum activity on polygalacturonic acid, 8.5%,55-70%, and 90% methylated pectin, respectively, providing evidence thatPelB is active on PGA as well as highly methylated pectin (FIG. 9).

Thermotoga maritime PelA showed highest activity on PGA, with only 41%and 2% activity on 30% and 74% methylated pectin, which differs from theactivity observed by P. amylolyticus C27 PelB. In addition, T. maritimePel A optima were pH 9.0, 90° C., and calcium was required for activity,making it the most thermoactive pectate lyase known to date (Kluskens,et al., 2003, Biochem. J, 370, 651-659).

Although the percentage of maximum activity for C27 PelB on 90%methlyated pectin is lower than that observed for C27 PelA, the specificactivities for PelA and PelB on the 90% methlyated pectin were similar.In addition, the specific activity for PelB on 8.5% methlyated pectin isalmost twice the specific activity observed for PelA. PelB shows moreenzymatic activity per protein concentration than PelA.

P. amylolyticus C27 PelA and PelB are not only the first pectate lyasesdescribed in P. amylolyticus, but also show an unusual combination ofpectate lyase and pectin lyase activity by degrading both highlymethylated pectin and polygalacturonic acid, respectively. Since bothenzymes require Ca²⁺ for activity, they are considered pecate lyasesinstead of pectin lyases. Pectin lyases do not require Ca²⁺ foractivity. This unusually activity has only been seen in two otherenzymes which are in the pectate lyase family III group: P.barcinonensis PelA and B. subtilis PelC.

In addition, P. amylolyticus C27 PelA is part of a subgroup of fivehomologous enzymes that are the only pectate lyases in family IIIproduced from nonpathogenic microorgaisms: P. barcinonensis pectatelyase A, B. subtilis pectate lyase C, Bacillus sp. P-2850 pectate lyase,Bacillus sp. KSM-P15 pectate lyase, and P. amylolyticus pectate A. Sowithin this subgroup are three enzymes that show, the unusualcombination of pectate lyase and pectin lyase activity.

On the other hand, P. amylolyticus C27 PelB shows highest amino acididentity of only 28% to Bacillus sp. YA-14 PelK, B. licheniformis ATCC14580 Pel, and B. subtilis reference strain 168 Pel. P. amylolyticus C27PelB shows unique activity on a broad range of pectic structures. PelBonly shows homology to polysaccharide lyase family I, but it is missingsome of the conserved regions for PL family I, and one of the three Pelconserved regions (Xiao, et al., 2007, Appl. Environ. Microbiol.:10:1-28), (Henrissat, et al., 1995, Plant Physiol., 107, 963-976). IfPelB is not part of polysaccharide lyase family I, then it could be partof a novel family of polysaccharide lyases.

Example 3

Degradation of sugar beet pulp and examination of oligogalacturonides.Precultures of E. coli carrying plasmid pEDH27 were grown in LB with 50mg L⁻¹ ampicillin overnight with shaking at 37° C. and inoculated intoLB with 5% dry wt L⁻¹ sugar beet pulp to OD₅₅₀ 0.5. Sugar beet pulpcultures were grown at 37° C. with shaking and samples were removedevery 24 h.

To quantify oligogalacturonides with a degree of polymerization (dp)less than 6, fermentation supernatant was diluted 1:3 in water andethanol was added to a final concentration of 11% (vol/vol). Thesolution was incubated with agitation for 16 h at 4° C. and thencentrifuged at 7500 g for 15 min. This supernatant was diluted andanalyzed at 235 nm (Spiro et al., 1993, Carbohydr. Res., 247:9-20) andcompared to the absorbance of fermentation supernatant preparationimmediately after inoculation.

Construction of E. coli JP27. Plasmid pEDH27 was transformed by heatshock into E. coli LY40A to construct strain JP27.

Sugar beet fermentations and analysis of ethanol production and reducedsugars. Fermentations were performed essentially as described previously(Doran et al., 2000, Appl. Biochem. Biotechnol., 84-86:141-152). Sugarbeet pulp dry weight was calculated using a Denver Instrument IR 35Moisture Analyzer (Denver, Colo.). In a blender, 10 g dry wt sugar beetpulp, 100 mL of 2×LB liquid media, and water to a final volume of 200 mLwere blended at full speed for 10 s and then autoclaved in a 500 mLfleaker; blending was necessary to reduce particle size as very lowfungal enzymes loads were used. The fleakers were placed in a water bathat 45° C. and mixed with magnetic stirrers. The pH was adjusted to 4.5using a Jenco 3671 pH controller (San Diego, Calif.). Spezyme CP(Genencor; Copenhagen, Denmark) and pectinase from Aspergillus niger(Novozymes; Franklinton, N.C.) were added to the fleaker atconcentrations of 0.5 FPU/g dry wt and 4 PGU/g dry wt, respectively.After 24 h, the pH was increased to 6.8 and the temperature wasdecreased to 35° C. and maintained throughout the fermentation.Appropriate antibiotics were added to each fleaker, and they wereinoculated to an OD₅₅₀ 1.0 with E. coli strains LY40A or JP27.Fermentations were run with samples collected every 24 h untilcompletion.

To quantify ethanol production, gas chromatography (GC) was performed;fermentation supernatant samples were filtered with a 0.22 μm filterprior to analysis. Ethanol concentrations were normalized to zero toaccount for ethanol added from antibiotic stocks. Reducing sugaranalysis was performed using the dinitrosalicylic acid assay method(Miller, 1959, Anal. Chem., 31:426-428).

Degradation of sugar beet pulp. To examine potential applications of theC27 PelA for saccharification in lignocellulose fermentations to fuelethanol, its ability to degrade pectin in sugar beet pulp was examined.E. coli DH5α carrying plasmid pEDH27 was grown in LB with 5% dry wt L⁻¹sugar beet pulp and samples were taken to measure short chainoligogalacturonides that would be produced from pectate lyase activity.As shown in FIG. 10, the amount oligogalacturonides with a degree ofpolymerization <7 dramatically increased over 72 h for the strainexpressing PelA, while E. coli DH5α pUC19 did not increase. As sugarbeet pulp pectin is typically 60% methylated (Sun and Hughes, 1998,Polymer J., 30:671-677), the ability of PelA to act on methylated pectinis desirable; the majority of described pectate lyases cannotsignificantly degrade pectin without added pectin methylesteraseactivity.

Sugar beet pulp fermentations with E. coli JP27. To better assess theapplicability of PelA in fuel ethanol production processes, pEDH27carrying pelA was added to ethanologen E. coli LY40A, a KO11 derivative.E. coli LY40A and JP27 were grown in sugar beet pulp using very lowfungal enzymes during saccharification; low ethanol yields were observedas expected. E. coli LY40A achieved a maximum ethanol yield of 1.79 gL⁻¹ ethanol by 24 h (FIG. 11). E. coli JP27, however, reached a maximumof 3.17 g L⁻¹ ethanol by 120 h, after displaying a lag between 24 h and48 h. Examination of reducing sugars shows that sugars liberated byfungal enzyme saccharification are consumed within 24 h for bothstrains. E. coli LY40A reaches its maximum ethanol production at 24 hbecause it is incapable of further lignocellulose degradation. E. coliJP27 also consumes the sugars released by fungal enzyme degradation by24 h; however, after a lag (which was also observed with E. coli DH5αpEDH27, FIG. 3) a small increase in the reducing sugar concentrationwith a concomitant increase in ethanol production is seen, demonstratingthe degradation of pectin to smaller oligogalacturonides and release offermentable sugars by PelA.

Example 4

PelB showed homology to family I pectate lyases (PL1), but not to anyother class. PelB showed highest identity of only 28% to Bacillus sp.YA-14 PelK, B. licheniformis ATCC 14580 Pel, and B. subtilis referencestrain 168 Pel. In addition, PelB showed lower identity to other pectatelyases: 27% to Thermotoga maritime PelA, and 26% to B. subtilis BS-2Pel, and B. amyloliquefaciens TB-2 Pel.

Other than PelB, all of the enzymes listed above contain all threeconserved calcium binding sites, all three conserved catalytic sites,and all six of the sites conserved in all thermostable PL1 pecate lyase.PelB contains the three conserved calcium binding sites, but does notcontain any of the conserved catalytlic sites, and only six of the sevensites conserved in all thermostable PL1 pecate lyase.

There are four highly conserved consecutive Asn ladder positions in allPels that help to stabilize the β bend that is structurally unique tothe parallel β helix that is present throughout polysaccharide families1, 3, 6, 9, and 19. The Asn ladder can be composed of Asn or amino acidsthat are able to act with similar function, such as, Cys, Gln, Thr, orSer. PelB only contain half of the conserved Asn ladders: Ser²¹⁹ andGln³⁰³. Whereas, all of the other enzymes being compared to PelB in thisstudy contain all four of the conserved Asn ladders. PelB has Pro²⁴⁸ andGly²⁷⁰, instead of the Asn found in the other enzymes.

In addition, PelB only has six of the ten invariant amino acids highlyconserved in the pectate lyase superfamily. PelB does contain invariantamino acids, Gly⁴⁷, Gly⁴⁸, Asp¹⁸², Trp¹⁹³, Asp¹⁹⁵, and H¹⁹⁶. All of theother enzymes listed above contain all ten of the invariant amino acidsexcept Thermotoga maritime PelA, which differs only at Val⁴⁰ (referenceto TmaPelA). PelB's Ser²⁵⁶, Val²⁵⁸, Lys²⁴⁴, Tyr³⁶ differ from theinvariant Arg, Pro, Thr, and Gly, respectively.

There are three pectate lyase conserved sequence patterns: vWIDH,AxDIKGxxxxVTxS, and VxxRxPxxRxGxxHxxxxN. PelB only contains two of thethree conserved sequence patterns. PelB and the enzymes mentioned aboveall contain the vWIDH sequence which is the core structure of theparallel β-helix. Trp, Asp, and His are three of the 10 invariant aminoacids found in all pecate lyases, and are Trp¹⁹³, Asp¹⁹⁵, and H¹⁹⁶.

The second sequence, AxDIKGxxxxVTxS, is found partially homologous inPelB and the other enzymes being compared to it. Asp²⁰⁶ of PelB is aninvariant amino acid that is seen in pectate lyases, but not in pectinlyases. It is thought to participate in the calcium coordination. Thisis not surprising since pectin lyases do not require calcium to beactive like pectate lyases do. Thr²¹⁵ and Ser²¹⁷ are also invariantamino acids that are seen in pecate lyases, but not in pectin lyases.They are located on the β strands near the vWIDH region.

The third sequence, VxxRxPxxRxGxxHxxxxN, is not found in PelB, but ispresent in the other enzymes being compared. The two Arg present in thissequence are positively charged amino acids near the calcium bindingsite that recognize the negatively charged substrates. The first Argpresent in this sequence is also one of the ten invariant amino acidsfound in the pectate lyase superfamily, but PelB has Ser²⁵⁶. The Pro isalso one of the ten invariant amino acids found in the pecate lyasesuperfamily, and is involved in the calcium binding by dictating theorientation of the first Arg in this sequence (PelB has Ser²⁵⁶ instead)to bind to water. The second Arg in the sequence is a conservedcatalytic site that is present in pectate lyase and absent in pectinlyases. PelB does not conserve this Arg and instead has Ser²⁶¹. The Hispresent in this sequence is also positively charged invariant residuefound in the Pel subfamily, but PelB does not conserve it and hasIle²⁶⁶.

In addition to the two arginines mentioned in the VXXRxPxxRxGxxHxxxxNsequence, the other catalytic binding site that is not conserved is apositively charged amino acid, Lys¹⁸⁹ (reference to TmaPelA). PelB has agap at this conserved location. These three amino acids, two Arg and oneLys, are positively charged amino acids that are located near thecalcium binding site and allow for the recognition of negatively chargedsubstrates. The presence of Ser²⁵⁶ and Ser²⁶¹ instead of the conservedArg and a gap instead of the conserved Lys could be the reason the C64PelB has activity on PGA and highly methylated pectin even though it isa pectate lyase.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-34. (canceled)
 35. An isolated polypeptide having pectinase activity,wherein the polypeptide comprises an amino acid sequence, wherein theamino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4 have at least 80% identity.
 36. The isolated polypeptide of claim35 wherein the isolated polypeptide is expressed by a geneticallymodified microbe.
 37. The isolated polypeptide of claim 36 wherein themicrobe is a gram-negative microbe or a fungus.
 38. The isolatedpolypeptide of claim 36 wherein the microbe is E. coli or S. cerevisiae.39. A composition comprising an enriched polypeptide having pectinaseactivity, wherein the polypeptide comprises an amino acid sequence,wherein the amino acid sequence and the amino acid sequence of SEQ IDNO:2 or SEQ ID NO:4 have at least 80% identity.
 40. A method fordegrading pectin comprising: contacting a composition comprising pectinwith a polypeptide having pectinase activity under conditions suitablefor the degradation of the pectin, wherein the polypeptide comprises anamino acid sequence, wherein the amino acid sequence and the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4 have at least 80% identity, andwherein the pectin is degraded.
 41. The method of claim 40 furthercomprising contacting the degraded pectin with a polypeptide havingoligogalacturonate activity.
 42. The method of claim 40 wherein thepolypeptide is expressed by a genetically modified microbe, and whereinthe contacting comprises contacting the pectin with the geneticallymodified microbe.
 43. The method of claim 42 wherein the geneticallymodified microbe expresses an exogenous polypeptide havingoligogalacturonate activity.
 44. The method of claim 42 wherein thegenetically modified microbe produces ethanol.
 45. The method of claim40 wherein the composition comprises a lignocellulosic material.
 46. Themethod of claim 45 wherein the lignocellulose material is obtained froma fruit or a vegetable.
 47. The method of claim 42 wherein the microbeis a gram-negative microbe or a fungus.
 48. The method of claim 47wherein the microbe is E. coli or S. cerevisiae.
 49. A method forproducing a metabolic product comprising: contacting a compositioncomprising pectin with a genetically modified microbe under conditionssuitable for the degradation of the pectin, wherein the geneticallymodified microbe comprises a polypeptide having pectinase activity,wherein the polypeptide comprises an amino acid sequence, wherein theamino acid sequence and the amino acid sequence of SEQ ID NO:2 or SEQ IDNO:4 have at least 80% identity, and wherein the genetically modifiedmicrobe produces a metabolic product.
 50. The method of claim 49 whereinthe metabolic product is ethanol.
 51. The method of claim 49 furthercomprising contacting the composition with a polypeptide havingoligogalacturonate activity.
 52. A method for producing a metabolicproduct comprising: contacting a composition comprising pectin with agenetically modified microbe under conditions suitable for thedegradation of the pectin, wherein the genetically modified microbecomprises an exogenous polypeptide having pectinase activity and anexogenous polypeptide having oligogalacturonate activity, and whereinthe genetically modified microbe produces a metabolic product.
 53. Themethod of claim 52 wherein the metabolic product is ethanol.
 54. Themethod of claim 52 wherein the microbe is a gram-negative microbe or afungus.